A Genetic Map Ofnon-Mendelian Genes in Chlamydlomonas* Ruth Sager and Zenta Ramanis

Proceedings of the National Academy of S&ienes Vol. 65, No. 3, pp. 593-600, March 1970

A Genetic Map ofNon-Mendelian Genes in Chlamydlomonas* Ruth Sager and Zenta Ramanis

DEPARTMENT OF BIOLOGICAL SCIENCES, HUNTER COLLEGE OF THE CITY UNIVERSITY OF NEW YORK Communicated by E. L. Tatum, December 22, 1969 Abstract. A group of eight non-Mendelian genes have been shown by recombination analysis to be linked into a linear structure or chromosome. Similar genetic maps of gene order and relative distances between genes have been constructed by two methods, one based on additivity of recombination frequencies, the other on frequency of reciprocal recombination with a postulated attachment point. The data indicate that the progeny are diploid for this linkage group, and that the strands are distributed in a precisely oriented manner at mitosis. Evidence is discussed in support of the view that this linkage group is located in chloroplast DNA.

In this paper we present evidence that a set of non-Mendelian genes in Chlamy- domonas can be mapped into a linear linkage group. The first non-Mendelian gene was described in 1908 by Carl Correns.1 Numerous examples of non-Men- delian heredity have been reported in the intervening sixty years,2 but this paper provides the first evidence that non-Mendelian genes are organized into chromosome-like structures that can be mapped by formal genetic methods. The first instance of recombination between two pairs of non-Mendelian genes was reported in 1963,3 the same year in which the presence of DNA in chloroplasts and mitochondria was established.4 Although knowledge of the presence of organelle DNA did not contribute directly to the genetic analysis reported here, correlations between the behavior of these genes and their DNA's will be of central importance in further studies. It is likely, as discussed below, that the linkage group described here is physically located in chloroplast DNA. The long lag in developing non-Mendelian genetics resulted in part from two obstacles: (1) the difficulty in obtaining non-Mendelian mutations, and (2) the maternal pattern in inheritance typical of non-Mendelian genes, which precludes recombination analysis and mapping. Methods for overcoming these obstacles were developed with Chlamydomonas.3 5 We found that cells suitably treated with streptomycin give rise to mutations of non-Mendelian genes but to virtually no mutations of nuclear genes.5 Despite its spectacular effectiveness, the molecular basis of this differential mutagenic action of streptomycin is not known. Maternal inheritance has been the key to the identification of non-Mendelian genes but has also been a serious impediment to their further study. In Chlamy- domonas we discovered that UV irradiation of the female parent immediately before mating would effectively block maternal inheritance under conditions of 593 Downloaded by guest on September 29, 2021 594 GENETICS: SAGER AND RAMANIS PROC. N. A. S.

little or no lethality.7 With UV irradiation we can routinely produce biparental progeny with non-Mendelian genomes from both parents. These progeny provide the material for recombination analysis. Materials and Methods. Mutants: The non-Mendelian genes ac1 and ac2 representing respectively leaky and stringent requirements for acetate were described before, as were sm2 (previously called sr, resistant to 500 ,g streptomycin per ml) and sm4 (previously called sd, streptomycin-dependent). Sm3 is a low-level streptomycin- resistant, nea is a neamine-resistant, obtained from N. R. Gillham who calls it nr, and ery is resistant to 50 og erythromycin per ml. Csd is a conditional mutant, requiring streptomycin to grow at 350C. Crosses: The studies to be reported here were carried out with the following crosses: Cross 1: 6978a (aC2+ acW sm3-s Sm2-scsd-s) X 7018g (ac,ac2 + sm3-r sm2-r csd-r) Cross 2: 11108-5 (ac2+ ery-r sm2-s) X 7018g (ac2 ery-s sm2-r) Cross 3: 229-3-4 (ac2+ ac( sm4-d ery-e sm2-s) X 11209-5 (ac2 aC + SM4-s ery-r sr2-r) Cross 4: 229-3-4 (ac1 sm4-d nea-s 8m2-8) X 12-6-2 (acl+ =f4-8 nea-r sm2-r) Cross 5: Z-12-4 (aC2+ ery-s nea-r smrs) X 11154-4 (aC2 ery-r nea-s sm2rr) Other crosses are listed in Table 3. Each parental pair also differs in three pairs of un- linked chromosomal genes (actidione resistance, methionine sulfoximine resistance, and mating type); these genes segregate in meiosis as previously described and provide a means for identifying the four zoospore products of meiosis. Crossing procedure: Gametes were prepared as previously described.6 Plus mating type (female) gametes received 40 sec of UV irradiation, and were then mated in the dark with unirradiated males. After 2 hr, the suspensions of newly formed zygotes were plated at suitable dilution, kept in light for 24 hr, and then incubated in the dark for 5 to 10 days. Synchronous germination was induced by exposure to light. After one mitotic doubling, clusters of 8 0 x octospores derived from single zygotes 0 were transferred to fresh plates where a PI l + P2 second doubling occurred. The cells were then respread so that the colonies derived from pairs of octospore daughter Zygote cells could be identified. By this pro- + cedure we obtained pedigrees of the first G3 o o two doublings of each zoospore, as 2 3 4 shown in Figure 1. Progeny cells were TetrasporesTetrospores grown to form colonies which were then 6' 6' 6' \b 6' \b classified for all markers.6' 7 la blb 2a \b2b 3a 3b 4a 4b Studies of closely linked genes: Octospores ' ' Results of cross 1 are shown in Tables 1 and 2. Zoospores were lol 1a2 fbl 0b2 2al 202 2bl 2b2 3al 3a2 3bl 3b2 4al 4a2 4b1 402 heterozygous for all four segregat- Octospore Daughters ing markers, as described in pre- FIG. 1.-Procedure for pedigree analysis. Ga- vious studies,6 8 and the appearance metes are mated and zygotes plated as noted in of pure homozygotes began at the the text. After germination, progeny of indi- first mitotic doubling. As shown in vidual zygotes are replated at the octospore stage and after one more doubling each pair of octo- Table 1, after two doublings 60-70 spore daughter cells is separated and allowed to per cent of the progeny were still form colonies. The 16 colonies, representing the heterozygous for one or both genetic first two doublings of each zoospore, are then classified for all segregating markers. regions. In a previous study of Downloaded by guest on September 29, 2021 VOL. 65, 1970 GENETICS: SAGER AND RAMANIS 595

TABLE 1. Segregation and recombination of closely linked genes (Cross 1). ac2+ aC sMr-s sm2-s X ac2 acl+ 8mrr smg-r A-Acetate Region Remaining Parental Segregants Recombinants hets* (%) (%) (%) ac2+ acl ac2 aci + ac2 + aci + ac2 + aci Total acg+ aci acgaci + aC2 + acg aci r Expt. 1 1419 13.2 10.4 2.1 74.4 Expt. 2 559 16.5 17.5 2.2 64.0 B-Streptomycin Region Remaining Parental Segregants Recombinants hets* (%) (%) (%) 8mg-s 8m2-8 8mg-r 8m2-r smg-fr 8mg2-s 8mg-8 8m-s Total 8mg-s 8m2-8 8msg- 8m2-r 8mg-r Sm2-8 8mg-r 8mg-r Expt. 1 1419 14.2 10.6 4.4 71.0 Expt. 2 559 21.2 18.6 2.5 57.8 Scored as per cent of total progeny after two mitotic doublings of zoospores. * May include additional genotypes which are phenotypically het, such as acg + ac +/ac2 acg and SM3- 8mg-rin/&M-f M2-8. TABLE 2. Linkage of acetate and streptomycin regions (Cross 1). Progeny class Recombination Numbers of Progeny$ 1 2 3 region Expt. 1 Expt. 2 Expt. 3 ac2+ ac1 Smg-s 8mg-s Parental 75 32 66 ac2 ac1 + smg-r sm2-r Parental 64 36 52 aC2+ ac1 smg-r smg-r 2 13 7 71 ac2 ace+ 8mgs siM2-s 2 20 15 57 acg+ ac1 smg-r smgs 2 + 3* 13 0 nst aeg ac1 + Smg-r s8M-s 3 15 6 ns ac2+ aci+ sn-r sm2-r 1 7 2 7 ac2+ aci+ 8mrs 8mg-s 1 + 2 5 3 8 ac2+ ac+smvr8smg-s 1 + 3 2 0 ns Total progeny segregated 214 101 261 Recombination in Region 1 6.5 5.0 5.8 2 23.9 25.0 52.2 3 14.0 6.0 ns * Position of 8mg- between acg and sn2 assigned on basis of data in Table 3 and unpublished. t ns = not scored. t Expt. 1 and 2 same as Table 1; Expt. 3 scored after 4 to 6 doublings. this cross8 we showed that the rate of segregation in these genetic regions is constant, with the surviving fraction of heterozygotes decreasing exponentially. No genetic class of persistent heterozygotes has been detected. In Table 1, the ratio of parental alleles among the progeny after two doublings appears to be close to 1:1 in both regions. The new classes ac+ ac2+ and sm3-r sm2-s are recombinants. The reciprocal recombinants are present but special methods are required to detect them, and they cannot be readily enumerated. The ac1-ac2 double mutant is phenotypically like ac2 and has been identified genetically.6 The sm3-s sm2-r recombinant is phenotypically like the double resistant; it has also been identified genetically.9 Linkage between acetate and streptomycin regions: The classes of progeny obtained in cross 1 have been listed in Table 2, together with the numbers of Downloaded by guest on September 29, 2021 596 GENETICS: SAGER AND RAMANIS PROC. N. A. S.

each class recovered in experiments 1 and 2 after two doublings, and in experiment 3, after four to six doublings of the zoospores. The linkage between the acetate and streptomycin regions clearly seen in experiments 1 and 2 has van- ished in experiment 3. This disappearance of linkage between loosely linked markers is evidence of the continuing recombination occurring in heterozygotes, and points up the importance of restricting the genetic analysis to the first two mitotic doublings after meiosis. Further evidence that the acetate and streptomycin regions are linked comes from data presented below showing that the genes sm4, nea, and ery are located between ac, and si2. Recombination frequencies in Table 2 were computed by considering only progeny which were segregated (i.e., homozygous) for all four markers. This method of computation is useful in testing for linkage, but neglects a sizeable fraction of the progeny in which some genes have segregated and others are still heterozygous. For example, only 14 ac+ recombinants were included in experiment 1, Table 2, and the frequency of ac+ was 6.5 per cent among the homozygous progeny. An additional 16 ac+ recombinants from progeny still heterozygous for sm, excluded from Table 2, were included in experiment 1, Table 1. Here the ac+ frequency was 2.1 per cent in the total population of progeny. The 2 per cent value is probably more accurate than 6.5 per cent since the computation method used in Table 1 is based on an unselected and larger sample than the method used in Table 2. We have therefore adopted the measure of recombinants in the total population as the preferred method of computing recombination fractions for most purposes. Linkage between nea and sM2: Linkage between nea and sm2 was examined in reciprocal crosses between double sensitive and double resistant strains. Similar recombination frequencies, 19.9 and 18.0, were found in the two crosses with 1200 progeny scored in one cross and 600 in the other. The relation between nea and sm2 is of particular interest, because Gillham reportedl' linkage between these markers in the cross nr ss X ns sr, but inde- pendent assortment in the cross nr sr X ns ss. He examined progeny from fully grown colonies (about 20 doublings), and in view of the scrambling which occurs, it is surprising that he found any linkage at all. The rate of disappearance of linkage probably depends on the relative rates of homozygosis vis-a-vis recombination among remaining heterozygotes. Mapping of markers by recombination analysis: The mutant genes smi (streptomycin-dependent) and ery (erythromycin-resistant) were crossed into various stocks in order to examine recombination frequencies in multiply-marked crosses. The results of three such crosses are summarized in Table 3. Detailed descriptions of these crosses will be published separately. For present purposes, the numbers of all detected recombinant types have been enumerated, and recombinant frequencies computed in the simplest manner, as a fraction of the total progeny scored. In each cross we found that the gene order could be established unequivocally by means of additivity of intervals; and the same order emerged in each cross. However, the numerical recombination fractions differed somewhat from one cross to another. For purposes of comparison, the recombination frequencies were normalized as noted in Table 3. Cross 3 was chosen as the standard because the five markers Downloaded by guest on September 29, 2021 VOL. 65, 1970 GENETICS: SAGER AND RAMANIS 597

TABLE 3. Computation of relative map distances. Number of _-Recombants*- Relative Cross Markers progeny (%) map distancet 1 ac2+ ac1 sm3rr sm2-r csd-s 1978t acr-ac, 2.1 2.7§ ac2 ac1 + am,-r am,.-r cad-r SmrSm2 3.9 5. 0§ 559t 8m2-ced 4.8 3.111 smr-csd 6.8 4.4 2 ac2 erY-rsm2-r 551 ac2-ery 6.4 12.0 ac2 ery-a smrr ery-sm2 4.9 9.25 ac2-sm2 6.7 12.6# 3 ac2+ ac37m4dery-a sm30 ac2-aCl 1.35 2.7§ aC2 ac+ 8m4-s ery-r sm2-r ac1-am4 4.3 4.3 aC2-Sm4 5.4 5.4 sm4-ery 5.1 5.1 ery-sM2 9.2 9.2 aci-ery 12.2 12.2 8m4-8m2 8.65 17.3 aCl-8M2 9.5 9*5¶ ac, sm4-d nea-asM2-8 360 al-8M4 2.5 5.6 aci + 8m4-s nea-r sm2-r SM4-nea 3.3 7.4 nea-am2 2.5 5.6 ac-nea 6.1 13.7 Sm4-Cm2 2.5 11.2§ * Number of recombinants/total progeny scored. t Normalization procedure: Cross 1-% recombination X 1.352.1 (cross(cross 1)3) acact Cross 2-% recombination X 1.3 ( 3) acr2-m2 11.3 (cross 2) Cross 4-% recombination X 18.6 (cross 3) ace-8m2 8.3 (cross 4) Only part of the experiment scored for cad. § Per cent recombination X 2 because only one of the two recombinant types was detected. | Unequal numbers of reciprocal recombinants. Low value because of long map interval.

segregating in the cross were well distributed across the linkage group. The data of crosses 2 and 4 were normalized by setting the map distance between the furthest markers equal to that found in cross 3. In cross 1, however, no inter- mediate markers were present between ac1 and sm3; and consequently the observed distance between them was very low. We therefore used the frequency of recombination between ac1 and ac2 as the basis for normalizing recombination fractions in this cross. By incorporating aci and ac2 routinely into crosses, it may be possible in the future to use the frequency of ac+ recombinants as a standard measure for comparing recombination frequencies from one cross to another. The position of sm3 and csd with respect to sm2 was determined not only by the additivity of intervals, but also by other evidence, in particular cosegregation frequencies.9 All recombination involving cad showed a great excess of the cad-a class for reasons as yet unknown, and further studies are in progress. Mapping of an "attachment point" (ap): Our method of pedigree analysis, in which we identify the pairs of sister cells resulting from the first and second doublings of each zoospore, provides detailed information on the segregation patterns of each gene pair. This information can be used for mapping purposes. Downloaded by guest on September 29, 2021 598 GENETICS: SAGER AND RAMANIS PROC. N. A. S.

As previously reported,8 both reciprocal and nonreciprocal events occur, classified as follows: Type I: both daughter cells heterozygous Type II: one daughter heterozygous; the other homozygous for either parental allele (nonreciprocal) Type III: both daughters homozygous, each for a different parental allele (reciprocal) When progeny segregating for several markers are classified in this way, one notes that cells may be homozygous for any number of markers from one to the complete set. Linked genes may segregate together in either a Type II or a Type III pattern, and the frequency distributions can be used to evaluate linkage and to construct maps. A comparison of data analyzed by segregation frequencies and by recombination will be presented elsewhere, except for the finding of polarity in Type III frequencies which will be discussed now. The Type III events may be examined for similarity to mitotic recombination of nuclear genes, by looking for evidence of polarity. In the nuclear system, chromosome distribution is governed by the centromere, a specialized region of the chromosome to which spindle fibers attach, pulling the sister chromatids to op- posite poles. The further a particular gene lies from its centromere, the more frequently will an exchange event occur between them. In heterozygotes, the exchange event may lead to homozygosis, and consequently, segregation frequencies can be used to measure gene-centromere distances, and to map the position of the centromere. We have used an analogous procedure to look for polarity by enumerating the number of Type III segregants for each gene. We found that a definite polarity does exist, which parallels the gene order shown in Figure 2a. The only excep- tions were in cross 5 where ery was beyond sm", and in cross 3, where the polarity between sin4 and ac2 was inverted. To compare the Type III frequencies with the recombination data of Table 3, the Type III data were normalized with respect to the map distance as shown in Table 4. The data of crosses 1 and 2 were expanded, assuming the distance from ac2 to s"2 20. On that basis the distance from the ap to ac2 is 15.6. In crosses 4 and 5 the relative distances between markers based upon Type III segregation frequencies were very similar to those in Figure 2a (except for ery in cross 5). How- ever, the position of all the markers relative to the ap was closer to ap than were those of crosses 1 and 2. The data of crosses 1, 2, 4, and 5 were combined into one map as shown in Figure 2b. It should be stressed that the map distances given in Figures 2a and b are only relative measures of exchange frequencies. Our observations are an under- estimate because we see only those recombinants which are also homozygotes. Furthermore, the mechanism of Type II segregation is still totally obscure. The fact that linear maps can be constructed so readily is particularly encouraging in view of all that is yet to be learned about this system. Discussion. The results presented in this paper demonstrate by formal genetic methods that a group of non-Mendelian genes in Chlamydomonas are organized in a linear structure, i.e., a linkage group or chromosome. The successful appli- cation of mapping procedures adapted from classical genetics provides strong Downloaded by guest on September 29, 2021 VOL. 65, 1970 GENETICS: SAGER AND RAMANIS 599

'7.3 1 12.2 l 9.2 cross 3 1 5.4| Op X2 27 CC1 Sm4 51 ery fce SIn3 sM2 csd 5.0 ~~~~3.1 4.4 cross 1 ~~~~~12.0~ ~ ~~ 9.25 Icross 2 5.6 I1 7.4 11 5.61 11.2 cross4 13.7 a

10.7 -1 9.3 - cross 2 op 6 cc2 OCc Sm4 fry nee Sm2 0-5_ISI I_ I__ 5.0 8.9 cross 4 13.9 8.0 4.3 cross 5 12.3

FIG. 2.-Genetic map of non-Mendelian genes. a, Based on recombination frequencies. b, Based on frequency of reciprocal recombination (Type III segregation). The numbers represent relative frequencies, normalized as shown in Tables 4 and 5 and discussed in the text. evidence that this non-Mendelian system is embodied in DNA and endowed with a mechanism (attachment point) to regulate its distribution at mitosis. These results support and complement our previous finding8 that zoospores from biparental zygotes are diploid for their non-Mendelian genome, and that after replication the distribution of this genome to daughter cells is oriented so that as a rule both daughters are themselves diploid. We previously suggested8 that segregation from the hybrid or heterozygous to the homozygous state might result from haploidization. However, the fact that some linked genes may be- TABLE 4. Polarity of recombination with attachment point (ap). Relative distance Cross* Gene % Type III from apt 1 aC2 3.9 15.6 Sm2 8.9 35.6 2 ac2 4.65 15.6 ery 7.9 26.3 8m2 10.7 35.6 3 ac2 16.2 ... sm4 10.8 ery 18.9 ... 4 ac1 10.0 18.0 Sm4 15.0 23.0 nea 23.9 31.9 5 ac1 10.0 18.0 nea 18.0 26.0 8m2 22.3 30.3 ery 30.0 ... * See Materials and Methods for description of crosses. t Crosses 1 and 2 adjusted to set acs-sm2 = 20 (cross 1 data X 4; cross 2 data X 2.15). Crosses 4 and 5 adjusted to map by moving all points 8 map units. Downloaded by guest on September 29, 2021 600 GENETICS: SAGER AND RAMANIS PROC. N. A. S.

come homozygous while others remain heterozygous, favors the view that cells remain diploid for this linkage group at all times so far observed. The linkage group described in this paper is probably located in chloroplast DNA, for the following reasons: (1) The only extra-nuclear DNA's known in Chlamydomonas are chloroplast and mitochondrial. There is 5-10 times as much chloroplast DNA as mitochondrial," suggesting that chloroplast genes may be more numerous and chloroplast mutations more frequent than mitochondrial ones. (2) In studies of the physical basis of maternal inheritance, we have found that chloroplast DNA from the male parent disappears in the zygote, coincident with the loss of non-Mendelian genes from the male.'2 These results demonstrate the maternal inheritance of chloroplast DNA per se. (3) The linkage group described in this paper is present in two copies,8 correlating with cyto- logical observations of two Feulgen positive regions in the chloroplast.'3 (4) Chlamydomonas contains but one chloroplast per cell and many mitochondria. It seems likely that mitochondrial genes will be present in several copies per cell, and will not show the diploid behavior described for this linkage group. The phenotypes of the markers in the linkage group, acetate-requirement and drug-resistance, might result from mutations of either chloroplast or mitochondrial DNA. In Chlamydomonas, chloroplast ribosomes are 70S14 and may be the site of action of streptomycin, neamine, and erythromycin. However, mitochondrial protein synthesis is also sensitive to these drugs."5 We are actively searching for an altered chloroplast protein resulting from mutation of one of these linked genes in order to localize this linkage group definitively. Our method of pedigree analysis has provided a wealth of data on segregation and recombination patterns, that is, exchange events affecting pairs of cells and their paired daughters. Beyond their use in mapping, the patterns provide the basis for developing and testing molecular models of the recombination process. This paper is dedicated with great admiration and affection to my teacher Professor MARCUS M. RHOADES. His pioneering studies of cytoplasmic inheritance aroused my enthusiasm for the subject and provided a model which I have sought to emulate. * This work was supported by USPHS grant GM-13970. 'Correns, C., Z. indukt. Abstamm.-Vererbungslehre, 1, 291 (1908). 2Jinks, J. L., Extrachromosomal Inheritance (Englewood Cliffs, New Jersey: Prentice-Hall, 1964). 3 Sager, R., and Z. Ramanis, these PROCEEDINGS, 50, 260 (1963). 4 Chun, E. H. L., M. H. Vaughan, and A. Rich, J. Mol. Biol., 7, 130 (1963); Sager, R., and M. R. Ishida, these PROCEEDINGS, 50, 725 (1963); Nass, M. M. K., and S. Nass, J. Cell Biol., 19, 593 (1963); Nass, S., and M. M. K. Nass, J. Cell Biol., 19, 613 (1963). Sager, R., these PROCEEDINGS, 48, 2018 (1962). 6 Sager, R., and Z. Ramanis, these PROCEEDINGS, 53, 1053 (1965). 7 Ibid., 58, 931 (1967). 8Ibid., 61, 324 (1968). 9 Sager, R., and Z. Ramanis, unpublished. 10 Gillham, N. W., these PROCEEDINGS, 54, 1560 (1965). 11 Sueoka, N., K. S. Chiang, and J. R. Kates, J. Mol. Biol., 25, 47 (1967); Lane, D., and R. Sager, unpublished. 12 Sager, R., and D. Lane, Federation Proc., 28, 347 (1969). 13 Ris, H., and W. Plaut, J. Cell Biol., 13, 383 (1962). 14Sager, R., and MI. G. Hamilton, Science, 157, 709 (1967). 16 Clark-Walker, G. D., and A. W. Linnane, J. Cell Biol., 34, 1 (1967). Downloaded by guest on September 29, 2021