Copyright 0 1990 by the Genetics Society of America

Genetic Instability of Clathrin-Deficient Strains of Saccharomyces cerevisiae

Sandra K. Lemmon,’ Carol Freund, Kathleen Conley and Elizabeth W. Jones Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213

Manuscript received February 15, 1989 Accepted for publication September 16, 1989

ABSTRACT Saccharomyces cerevisiae strains carrying a mutation in the clathrin heavy chain gene ( C H C I ) are

genetically unstable and give rise to heterogeneous populations of cells. Manifestations of the instability include increases in genome copy number as well as compensatory genetic changes that allow better growing clathrin-deficient cells to take over the population. Increases in genome copy number appear to result from changes in ploidy as well as alterations in normal nuclear number. Genetic background influences the frequency at which cells with increased genome content are observed in different Chc- strains. We cannot distinguish whether genetic background affects the rate at which aberrant nuclear division events occur or a growth advantage of cells with increased nuclear and/or genome content. However, survival of chcl-A cells does not require an increase in genome copy number. The clathrin heavy chain gene was mapped 1-2 cM distal to K E X l on the left arm of chromosome VZZ by making use of integrated 2r plasmid sequences to destabilize distal chromosome segments and allow ordering of the genes.

C LATHRIN-coated membranes and coated vesi- cles (CV) are involved in vesicular transport in

the endocytic and secretory pathways of eukaryotic cells (PEARSE and BRETSCHER 1981; BRODSKY 1988). In animal cells it is well established that CV play an important role in receptor-mediated endocytosis (GOLDSTEIN et al. 1985) and in transport of certain molecules as they exit the Golgi (GRIFFITHS and SI- MONS 1986). The major subunit of the polyhedral lattice that surrounds these vesicles is clathrin, a pin- wheel-like molecule (UNCEWICKEL and BRANTON 198 1) whose arms are composed of three heavy chains and three light chains (PEARSE 1976; KIRCHHAUSEN and HARRISION 198 1).

In order to learn more about the structure and function of coated vesicles, we and others undertook an investigation of clathrin in Saccharomyces cerevisiae (MUELLER and BRANTON 1984; PAYNE and SCHEKMAN 1985; LEMMON and JONES 1987). The yeast clathrin heavy chain gene (CHCl ) was cloned and genetic studies were initiated to determine the consequences of generating a null mutation in CHCl (PAYNE and SCHEKMAN 1985; LEMMON and JONES 1987). The results of such experiments showed that yeast cells can survive, albeit poorly, without clathrin heavy chains in some genetic backgrounds, but in other back- grounds they cannot. The ability to survive a clathrin null mutation in our strains was attributed to an

’ Current address: Department of Molecular Biology and Microbiology,

44 106. Case Western Reserve University School of Medicine, Cleveland, Ohio

The publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. $1734 solely to indicate this fact.

Genetics 124 27-38 (January, 1990)

independently segregating gene, which we have termed suppressor of clathrin deficiency ( scd l ) (LEM- MON and JONES 1987). If the nonsuppressing allele ( S C D l ) of this locus is present, cells lacking clathrin heavy chains are inviable.

In the process of characterizing Chc- cells and testing for the presence of the suppressor allele, we observed that chcl-A scdl cells are genetically unstable (LEMMON and JONES 1987). First, we noticed from segregational analysis of crosses that Chc- strains fre- quently gave polyploid zygotes. In the one case ex- amined in most detail, the zygote was tetraploid and contained three copies of the genome of the Chc- parent, and one copy of the genome from the Chc+ parent. Microscopic examination of cells stained with 4’,6’-diamidino-2-phenylindole (DAPI) revealed that Chc- cells occasionally show aberrant nuclear divi- sions, resulting in mother cells with two nuclei (LEM- MON and JONES 1987). Since endomitosis by itself cannot yield a triploid nucleus, it seems necessary to postulate that an abnormal nuclear fusion event oc- curred either within the Chc- cell or in the zygote, possibly involving the fusion of nuclei from a binu- cleate (but 3n total) Chc- cell. Second, although chcl- A scdl cells show a slow and temperature sensitive growth on a variety of media and many dead or lysing cells are observed in cultures, these properties vary widely from isolate to isolate and poorly growing isolates often give rise to faster growing but still Chc- derivatives.

Here we report on our analysis of this genetic instability and our attempts to begin to understand the causes of the growth heterogeneity and the com-

28 S. K. Lemmon et al.

TABLE 1

Saccharomyces cerevisiae strains used in this study

Number Genotype” Notes, Source or Referenceb

BJ2649 BJ265 1 BJ2665 852738 BJ2744 BJ2746 BJ2752 BJ2754 BJ2755 BJ2757 BJ2990 BJ3068

BJ3083 BJ3118 BJ3119

MATa lys2 trp5 Can’ M A T a lys2 trp5 Can‘ MATa ura3-52 leu2 MATa CHCl leu2 ura3-52 trpl scdl MATa leu2-3 pet14 argl lys7 met6 ade3 His- MATa leu2-3 trpl met4 aro7 his3 lys l l SUC2 MAL3 MATa leu2-3 met13 ade2 cdc4 pet3 ura4 his4-15 lys2 MATa leu2-3 met14 ade5 pet8 ura3 his7 lysl MATa leu2-3 pha2 petX prtl arg8 MATa leu2-3 met14 ade5 pet8 ura3 his7 lysl MATa CHCl his3 trpl ura3-52 cir” MATa CHCl leu2 ura3-52 trpl hisl ade6 scdl MATa CHCl leu2 URA3 TRPl HISl ADE6 SCDl MATa CHCl::2p,URA3 his3 trpl ura3-52 cir” MATa ura3-52 lys2 Ade- trpl-A101

”” -”

MATa chcl-A20::LEU2 leu2 ura3-52 trpl hisl ade6 scdl MATa CHCl leu2 URA3 TRPl HISl ADE6 SCDl ” ”-

Spores from BJ3 1 19 C H C l ( 4 A , 14A, 18C, 19A, 20B, 29D, 32C, 34A, 35A, 35D)

MATa chcl-A::LEUZ leu2 URA3 trpl hisl ade6 scdl (24B) MATa CHCl leu2 ura3-52 TRPl HISl ADE6 (35D)

chc1-A::LEUZ scdl (4D, 9B, 17C, 24B, 24D, 29C, 34C)

BJ3131 . .

MATa CHCl leu2 ura3-52 trpl hisl ade6 scdl MATa CHCl leu2 ura3-52 TRPl HISl ADE6 SCDl ”” ”-

BJ3 173 MATa CHCl ura3-52 ade2-101 trpl-A101 lys2-801 BJ3 174 MATa CHCl ura3-52 ade2-101 trpl-A101 leu2-A1 lys2-801 BJ333 1 MATa chcl-A20::LEU2 leu2 ura3-52 trpl hisl ade6 scdl

MATa CHCl leu2 ura3-52 TRPl HISl ADE6 SCDl Spores from BJ333 1

” ”- [PSLGI

chcl-A::LEUZ scdl (2A, 3D) chcl-A::LEU2 scdl [PSLG] (13A)

BJ3332 MATa trpl hisl ura3 M A T a T R P l H I S l U R A 3

K- R-

BJ3369 MATa thrl ade2 kexl-1 [KIL-kl] B13371 MATa hisl ade2 kexl-1 [KIL-kll

- ”

G3378 MATa chcl-APl::LEU2 ieu2 ura3-52 trpl hisl ade6 scdl MATa CHCl leu2 ura3-52 TRPl HISl ADE6 SCDl ” ”- [ P S W

Spores from BJ3378 chcl-A::LEU2 scdl (8C, 25C) chcl-A::LEU2 [pSL6] (lB, lC, 3D, 4A) CHCl [pSL6] (1 A, lD, 3C,4B)

BJ3379 MATa-~hcl-A22::LEU2 leu2 ura3-52 trpl hisl ade6 scdl MATa CHCl leu2 ura3-52 TRPl HISl ADE6 SCDl ” ”- [PSL61

Spores from BJ3379 chcl-A::LEUZ scdl (2D, 13B) chcl-AzLEU2 [pSL6] (lD, 2B, 3D, 4D) CHCl [pSL6] ( lB, 2A, 3B, 4C)

BJ3398 MATa leu2-3 ade5 ura3 met14 lysl thrl pet8 kexl-1 [KIL-k~] BJ3444 MATa CHCl::LEU2 leu2 ura3-52 trpl scdl BJ3445 MATa CHCl ura3-52 ade2-101 trpl-A101 leu2-A1 lys2-801 Lys+ BJ3446 MATa CHCl ura3-52 his3-A200 trpl-A101 leu2-A1 lys2-801 BJ3447 MATa CHCl ura3-52 his3-A200 trpl-A101 leu2-A1 lys2-801 Lys’ BJ3448 MATa CHCl ura3-52 ade2-101 trpl-A101 leu2-A1 lys2-801 BJ3449 BJ3445 X BJ3446 BJ3450 BJ3447 X BJ3448 BJ3491 MATa leu2 ade5 ura3 lysl trpl kexl-1 [KIL-kl] BJ3539 MATa chcl-A40::LEU2 leu2-A1 ura3-52 trpl-A101 his3-A200

MATa CHCl leu2-A1 ura3-52 trpl-A101 HIS3 ”

ade2-101 lys2-801 Lys+ [pSL6]

A D E 2 1 ~ ~ 2 - 8 0 1 Spores from B13539

A343-IA, GABER et al. (1983) A236-57B, GABER et al. (1983) A193-16C, GABER et al. (1983) A121-3A, GABER et al. (1983) A334-49B, GABER et al. (1983) A121-3D, CABER et al. (1983) DBY703, C. FALCO -d , LEMMON and JONES (1987)

YP8 1, P. HIETER -d , LEMMON and JONES (1987)

YP54, P. HIETER YP98, P. HIETER

-d./

- 5x47. R. WICKNER

92A, R. WICKNER 93, R. WICKNER

-da

- -

(24A from 3369 X 2757)

h -

h -

(39A from 3398 X 3444) A,‘

chcl-A::LELI2 (lB, 2C)

Clathrin-Deficient Yeast 29

Number Genotvoe“ Notes. Source or Reference’

BJ3543 MATa chcl-A39::LEU2 leu2-A1 ura3-52 trpl-A101 his3-A200 MATa CHCl leu2-A1 ura3-52 trpl-A101 HIS3 ”

ade2-I01 lys2-801 ADE2 1 ~ ~ 2 - 8 0 1

Lys+ [pSL6]

Spores from BJ3543 chcl-A::LEU2 (IA, lD, 2C, 5B, 5C)

CHCl or CHCl [pSL6] (IB, IC, 2A, 2D, 6B) MATa leu2-3,112 kexl::LEU2 ura3-52 adel [KIL-kl] Sc25K-13, DMOCHOWSKA et al. (1987)

chcl-A::LEUP [pSL6] (6C) -

BJ3710 BJ3902 MATa leu2 ade5 adel ura3 kexl::LEU2 [KIL-kl] (4A from 2754 X 3710) BJ39 16 MATa leu2 ade5 met14 ura3 pet8 kexl::LEU2 [KIL-kl] (ID from 2754 X 3710) BJ4203 MATa ade6 hisl Can‘ BJ4204 MATa ade6 hisl Can‘ BJ4469 MATa leu2 ura3 ADE5 CHCl::2p,URA3 KEXl trpl [KIL-kl] (1D from 3083 X 3902) BJ4470 MATa leu2 ura3 ADE5 CHCl::2p,URA3 KEXl trpl his7 [KIL-kl] (8B from 3083 X 3902)

-

- -

a SCDl or scdl is indicated only where known or inferred. chcl-A20, A21, A22, and A40 have LEU2 integrated in the “a” orientation, A39 has LEU2 integrated in the “b” orientation (see MATERIALS AND METHODS). In most cases, only the relevant heavy chain genotype is indicated for spores. Haploid genotypes of Chc- strains do not reflect changes in genome content.

Except where indicated, strains are from this study or this laboratory. ‘ BJ2649, BJ2651, BJ4203 and BJ4204 were derived from Cans strains of the same genotype, by selection for mutation to Can’ on

canavanine plates. It is presumed that these are can1 alleles. BJ3068 was the parent of BJ3119, BJ3131, BJ3331, BJ3378 and BJ3379. These spores from chcl-A::LEU2 heterozygotes should be haploid, since they have always carried a wild type clathrin heavy chain. ’ BJ3 13 1, which is homozygous for ura3, was obtained by selecting a FOA-resistant derivative of BJ3068 (BOEKE, LACROUTE and FINK

1984). BJ333 1 is a pSL6 transformant of a ura3-52/ura3-52 derivative of BJ3119 obtained by the FOA selection procedure. BJ3131 was transformed with pSL6 and then gene transplacement performed to generate two independent disruptants, BJ3378 and

BJ3379. BJ3449 and BJ3450 were transformed with pSL6 and then disruption performed, generating BJ3543 and BJ3539 respectively. The diploid parent of these strains was originally lys2-801/lys2-801, but, inadvertently, a Lys’ derivative was selected. Segregational

analysis has shown that the gene conferring Lys+ is an extragenic suppressor. ‘ A spore clone derived from a cross of a strain from our collection (BJ2665) to one from the collection of P. HIETER (BJ3173) was

backcrossed to a second strain from the Hieter collection (BJ3174). Intercrosses among the progeny of this latter cross produced the transformation recipients (BJ3449 = BJ3445 X BJ3446; BJ3450 = BJ3447 X BJ3448). Approximately 75% of the genetic contribution of the input haploids derived from the Hieter collection. All of these strains are supposed to be congenic to X2180-1B. Segregational analysis indicates that these strains are homozygous for a gene that allows growth of chcl-A cells. Whether the gene is scdl or another was not determined.

pensatory alterations in clathrin-deficient cells. In ad- dition, we present results of the mapping of CHCl adjacent to K E X l on the left arm of chromosome VZZ using a novel procedure to order the genes relative to the centromere.

MATERIALS AND METHODS

Materials: Enzymes were purchased from commercial sources and used according to the manufacturer’s instruc- tions. [a”P]dCTP and the random primer labeling kit were purchased from New England Nuclear Corp. Media supplies were purchased from Difco or premixed powdered batches of YEPD and LB were obtained from GIBCO/BRL. Glu- sulase or a comparable preparation were from Endo Labo- ratories or Sigma Chemical Co., respectively. L-Canavanine sulfate was from Sigma. Methylene blue was from Fisher Scientific.

Media: YEPD and synthetic media for yeast cultures were prepared and used as described previously (JONES and LAM 1973) . For YEPG plates, 5% glycerol replaced the glucose in YEPD. Canavanine medium was arginine omission syn-

thetic medium with 60 mg/liter canavanine sulfate added. Sporulation media (PSP and KAc) were those described in ZUBENKO and JONES (1 98 1) and JONES (1 972) respectively. Methylene blue plates were prepared as described in SOM- MERS and BEVAN (1 969). 5-Fluoro-orotic acid (FOA) plates were made according to the method of BOEKE, LACROUTE and FINK (1 984).

Strains: Yeast strains used in this study are listed in Table 1. Except for those used in chromosome mapping, all strains were derived from X2180-1B (MATa g a l 2 SUC2) or from crosses between strains in our isogenic series and strains congenic to X2180-1B. Throughout these studies, indi- cated genotypes of chcl-A scdl strains derived from chcl-A/ CHCl heterozygous diploids are the expected haploid gen- otypes and do not reflect possible increases in genome copy number.

Genetic methods: Procedures for mating, sporulation, dissection and scoring of nutritional markers were as de- scribed by MORTIMER and HAWTHORNE (1 969). All matings of chcl-A strains were made with populations from the original chcl spore clone rather than from selected single colonies except where indicated. Products of matings were selected as prototrophic colonies.

30 S. K. Lemmon et al.

The segregation of KEXl, which encodes a protease in- volved in processing of killer toxin and a-factor to their mature forms (DMOCHOWSKA et al. 1987), was scored essen- tially as described by SOMMERS and BEVAN (1969). Intially strains to be tested for killer function were replicated from a YEPD master directly onto a methylene blue plate con- taining a killer sensitive strain (BJ3332) and halos were scored after 1-2 days of growth at 30". Often the M1 double stranded RNA (KIL-kl) carrying the gene for killer toxin was not maintained. Therefore KIL-kl was reintro- duced by mating strains to be tested with MATa hex1 (BJ3369) and MATa hex1 (BJ3371) strains bearing KIL-kl and prototrophic diploids were scored by the halo assay.

To order CHCl and KEXl relative to the centromere, the diploid strain (BJ4469 X BJ3916) was inoculated into YEPD broth, grown overnight, diluted approximately 1000- fold into fresh YEPD and grown overnight again to allow for accumulation of cells that had undergone arm loss due to the integrated 2~ sequences on chromosome V I . Cells were spread on YEPD for single colony isolation and, after growth, were replicated to plates lacking adenine, leucine or uracil. BJ4470 X BJ3916 was streaked onto a FOA plate and Ura- segregants were selected. For both crosses, Ade- Ura- Leu+ segregants were subjected to random spore analysis after sporulation for 5-7 days on KAc plates. Sus- pensions of sporulated cells in 0.2 ml H20 with 10 PI glusu- lase were incubated at 30" for 1 hr and sonicated 3 X 30 sec using a Branson sonifier with a microtip probe at -80 W. After single colony isolation, colonies were scored for markers. Very few nonmaters, i.e., nonsporulated diploids, survived this regimen.

Plasmids: YCp50-CHCl (pSL6) (LEMMON and JONES 1987) contains the complete clathrin heavy chain gene in the plasmid YCp50 (KUO and CAMPBELL 1983), which car- ries URA3 as a selectable marker. pchcl-Aa::LEUZ (pSL12) and pchcl-Ab::LEUZ (pSL13) (LEMMON and JONES 1987), used for gene transplacement (ROTHSTEIN 1983), are pBR322 based plasmids containing sequences from the CHCl locus with the LEU2 gene inserted in place of the 4.9- kbBglII-BamHI CHCl fragment in the same (Aa) or opposite (Ab) transcriptional direction as CHCl. pSL5 (Figure 4A) was identified in a YEp24 yeast genomic DNA bank (CARL- SON and BOTSTEIN 1982) by colony hybridization (GERGEN, STERN and WENSINK 1979) using the 0.9-kb EcoRI fragment containing the 5' region of the heavy chain gene as a probe. PAD 17 (DMOCHOWSKA et al. 1987) is a YCp50 based plasmid that contains the KEXl gene and was obtained from A. COOPER and H. BUSSEY, McGill University.

Integration at the heavy chain locus: Gene disruptions of CHCl homozygous diploids were performed as described previously (LEMMON and JONES 1987). Briefly, pSL12 or pSLl3 DNA was digested with HindIII, generating a frag- ment containing the LEU2 gene flanked by sequences ho- mologous to CHCl. The product DNA was used to trans- form Leu- yeast cells, with selection for Leu+. Integration at the CHCl locus in all cases described herein was con- firmed by DNA blot analysis. For diploids that carried pSL6 (BJ3378, BJ3379, BJ3539, BJ3543), plasmid loss was used to confirm that the integration of the LEU2 gene occurred at chromosomal rather than plasmid sequences. Ura- segre- gants of such transformants remained Leu+. Strain BJ3444, which was used in mapping experiments, was generated by transforming BJ2738 with pSL12 that had been linearized at the unique NruI site residing -0.65 kb upstream of the 5' BglII site in CHCl. This integration generated a LEU2 disrupted version of CHCl adjacent to the intact gene. Strain BJ3083 was generated by transforming BJ2990 with pSL5 that had been linearized at the unique BstEII site in the

insert. This integration placed 2~ and URA3 sequences between CHCl and KEXl and is outlined in Figure 4A.

Microscopic observation: Cells were grown in YEPD to A600 of 1-5. Approximately five A600 units of cells were pelleted and resuspended in 0.5 ml YEPD for observation. For staining with methylene blue to reveal lysed or dead cells, yeast were washed and resuspended in phosphate- buffered saline (PBS) (DULBECCO and VOGT 1954) contain- ing 2% glucose. Just prior to observation, one-tenth volume of 0.3% methylene blue was added. Methylene blue staining was stable in the glucose/PBS solution so that gradual uptake by viable cells (both Chc' and Chc-) was minimal during the time of observation. Cells in suspension were applied to dry slides that had been precoated with poly-L-lysine or concan- avalin A. Excess liquid was removed and coverslips were applied to slides prior to observation with a light microscope equipped with Nomarski optics.

Tests for polyploidy by canavanine resistance: To di- rectly distinguish haploid cells from those of higher genome content, strains were screened for the frequency of UV- induced mutation to canavanine resistance (SCHILD, ANAN- THASWAMY and MORTIMER 1981). In the direct test, chcl-A strains were transformed to Chc+ with pSL6 and Ura+ transformants were selected. Ura+ isolates were patched onto YEPD masters with appropriate Cans haploid and diploid controls. After 2 days these were replicated to can- avanine plates and to synthetic complete plates as a control for growth, the plates were UV irradiated for 30 sec at 36 cm from a GE 30 W germicidal lamp (G30T8). The fre- quency of papillation to Can' was scored after 4 days of incubation at 30".

The indirect Can' test for increased genome number involved mating each chcl-A strain to a haploid Chc+ Can' strain of the opposite mating type (BJ2649, BJ265 1, BJ4203 or BJ4204), followed by a test for the ability to give Can' derivatives by mitotic recombination. Mass matings to Can' strains were performed with the population of Chc- cells grown up from the original spore clone and prototrophs were selected on minimal medium (MV). Approximately 32 individual prototrophs from each mass mating were patched onto master YEPD plates along with appropriate diploid controls. Care was taken to choose colonies of all different sizes from the MV plate to avoid selection of cells of a particular "ploidy."2 This resulted in a slight enrichment for petites among the smaller colonies selected. Masters were grown for 2 days and then replica plated to canavanine, to synthetic complete as a growth control, and to YEPG to test for petites. Since papillation to Can' could result from mitotic recombination, no UV irradiation was required. After 3-4 days of growth at 30", patches were scored for papillation and compared to Can'/Can" and Cans/Cans dip- loid controls. Petites did not display papillation to Can' after the usual period of growth, so such prototrophs were ex- cluded from the data analysis.

Other methods: The blot of chromosomes from YP81 (BJ3118) separated by orthogonal field alternation gel elec- trophoresis (OFAGE) (CARLE and OLSON 1985) was the generous gift of PHILIP HIETER (Johns Hopkins University). DNA probes were labeled with [aS2P]dCTP by nick trans- lation (MANIATIS, FRITSCH and SAMBROOK 1982) or the random primer labeling procedure (FEINBERG and VOGEL- STEIN 1983). Yeast transformation was by the method of ITO et al. (1983). Methods of cloning and routine bacterial

"Polyploidy" or "ploidy" refer to increases in genome copy number and do not distinguish polyploid cells from multinucleate cells. Polyploid or ploidy refer to increased genome copies within a single nucleus.

Clathrin-Deficient Yeast 31

TABLE 2

Observation of ”Polyploidy” in Crosses

Matings”

Chc- X Chc+ Germination* Segregation‘

A. Crosses of Chc- spores from BJ3 119 to Chc+ strains 4D X 35A 4D X Culb.d + 4n‘ 4D X 4.4 + 24n 9B X Culb.d - 17C X 32C + 24n 17C X 18C + 24B X 20B

24n

24B X 19A + 2n 24B X 35D + 4n‘ 24D X 32C 29C X 14A 29C X 29D + 24n 34C X 35D - 34C X 34A + 24n

-

-

- -

B. Crosses of Chc- to Chc+ spores from BJ3543 1A X 1B + 2n 1A X 1C + 2n ID X 1C + 2n 2C X 6B + 2n 5B X 2A + 2n 5B X 6B + 2n 5C X 2A + 2n 5C X 6B + 2n

a Matings in A were between chcl-A scdl spore clones (Chc-) and CHCI spore clones (Chc+) derived from tetrad analysis of BJ3119 except where noted.

* +, normal spore viability; -, few or no viable spores. ‘ 271, diploid segregation; 24n, tetraploid or pentaploid segrega-

tion. Culb. indicates that the Chc- spore clone was mated to five

strains (BJ2744, BJ2746, BJ2752, BJ2754, BJ2755) from the map- ping set from M. CULBERTSON (CABER et al. 1983).

e For 4D X Culb., tetrad analysis was performed on two chcl-A heterozygous non-mating spores each from crosses of 4D to BJ2746, BJ2752, and BJ2754. Results indicated that the 4D X Culb. zygotes were 4n. For 24B X 35D, tetrads from five non-mating chcl-A heterozygous spores were dissected and indicated that the 24B X 35D zygote was 4n.

manipulations were essentially those described in MANIATIS, FRITSCH and SAMBROOK (1982).

RESULTS

Observation of “polyploidy” in crosses: “Poly- ploidy” (see footnote 2) in cells carrying a null muta- tion at CHCI was first inferred to occur when chcl-A scdl spore clones derived from a chcl-AlCHC1 scdl l SCDl diploid (BJ3119) were mated to CHCI haploid strains. Tetrad analysis involving crosses of eight dif- ferent Chc- spore clones led to a spectrum of out- comes, a sampling of which is shown in Table 2A.

Many zygotes (12126) yielded few or no viable spores, a result we interpret to indicate triploid seg- regation. Thirteen of 14 crosses that resulted in viable spores showed segregation patterns expected if an effectively triploid or tetraploid Chc- cell had mated to a haploid Chc+ cell to give a tetraploid (4n) or

pentaploid (5n) zygote. (We could infer only the num- ber of genomes, rather than the number of nuclei, donated by the Chc- cell.) For example, instead of 2:2 segregation of markers in tetrads, virtually all spores were Leu+ whether they were Chc+ or Chc-, even though LEU2 marked only the chromosome carrying the deleted form of CHCI; recessive markers that entered the cross from the Chc+ parent were rarely observed in spore progeny; and mating type showed aberrant segregation, with most tetrads yielding two nonmating spores and two spores with the mating type of the Chc- parent. For four crosses (24B X 35D, 4D X 2746, 4D X 2752, 4D X 2754) (Table 2A), we examined meiotic progeny resulting from sporulation of two or more Chc+ nonmating spores and confirmed that the zygote parents were actually 4n rather than >4n and had yielded diploid spores. Such nonmaters, upon sporulation and tetrad analysis, gave a segrega- tion pattern expected for chcl heterozygous diploids. For 4D X 2746, a total of seven chromsomes were unambiguously found to be present in two copies in the diploid spore progeny; for 24B X 35D, three chromosomes were so monitored. An example of the tetrad data from a 4n zygote has been presented previously (24B X 35D) (LEMMON and JONES 1987). Because Chc- diploids do not sporulate, it was not possible to carry out a comparable analysis on these diploid spore types.

Only one of the 14 crosses that showed normal spore viability yielded a diploid (2n) segregation pat- tern and haploid progeny spores (24B X 19A). This result was our first indication that the mechanism by which scdl could prevent lethality of Chc- cells was not dependent upon an increase in ploidy or nuclear number. Another observation made from these crosses was that the population of Chc- cells from a given spore clone was heterogeneous, since several of them (4D, 24B, 29C, 34C) gave more than one out- come when crossed to Chc+ strains and 24B yielded all three outcomes (few viable progeny, 4n and 2n segregation).

Canavanine resistance test for “polyploidy”: The occurrence of “polyploidy” in chcl-A scdl cell lines was further examined by testing for their ability to mutate to Can‘ after UV irradiation. Since CANIS is dominant to canl‘, haploids should show a much higher frequency of papillation to Can‘ than cells of higher ploidy (or higher nuclear content). Initially we attempted to test for papillation of chcl-A scdl cells directly on canavanine plates after irradiation; how- ever, no chcl strains gave papillae on this medium. Since Chc- cells grow poorly on synthetic media, it was possible that chcl cells would appear canavanine sensitive regardless of their CAN1 genotype. There- fore, we transformed the Chc- cells to Chc+ with pSL6 and examined the ability of these transformants to

32 S. K. Lemmon et al.

I.'IC:CRE 1 .-(hwvaIlinc rcsistancc tc'st of "polvploidy'' h r (hc - cells trallsfnrmetl t o <:hc'. \laster plates ol'strains growl on YEPD were replicated t o cmawnine (upper panel) and synthetic complete (lower p;tnel) and U\' irr;ltli;ltctl a s described i n MATERIAIS AND METHODS. Plates were photographed after 4 days of growth at 30". Strains: rnw 1 A, r h r l - A [pSL.fi] (BJS379-I D, -2B. -3D and -41)); row 2A. C H C I [pS1.6] (BJ3379-IB. -!!A, -3B and -4C): row 3A. rhrI-A/ C H C I [pSI.fi] ( I and 2 are BJ3.778: 3 and 4 are Bj3379): row 4A. C I f C I / C H C I [pS1,6] ( a l l are 1!]333 1). Strains shown in rows I A and 2A were oht;linetl directly fl-onl diploids that carried the C H C I plasmid [pSLfi]. Kows 113-413: each row sllows four independent transformlnts from a given parent rhrl-A srdl strain after transfnr- I n a t ion to Chc+ w i t h pSI.6. The Chc- strains prior to transformation for each row were: row I B, B.JJJ78-XC: row 'LR, RJ3378-95C: row 3B, BJ3379-2D: row 4B. I3J3379-13B. Also ;Inalyzed (nor shown) ~vere: rhrl-A [pSL.6] spore clones BJ3378-lB. -IC, -3D and -4A; C H C I [pSI.fil spore clones BJ.7378-I A , - 1 11. -3C and -4B: ;Ind rhcl - A s rd l spores clones transformed to Chc' wi th pSL6: .?A and 34D frorn 13J3 1 19: !!A and 31) fronl q 3 3 3 I .

papillate to Can". Eight different ura3-52 chcl-A scdl strains (derived from three independent chcl-AICHCI

diploids) were transformed with the plasmid and four independent Ura+ Chc" transformants of each were tested for the ability to mutate to Can" (Figure 1).

The chcl-A scdl strains that had been transformed to Chc' (32/32) gave few or n o papillae on canavanine plates (upper panel, rows IB-4B) as compared to hap- loid controls (chcl -A [pSL6] derived directly from dissection of chcl-A heterozygotes carrying the plas- mid (row 1.4) or CHCI [pSL6] (row 2A)). Diploids heterozygous or honlozygous for CHCI showed little papillation, as expected (rows 3A and 4A). T w o Chc+ transformants (row 3B, patches 2 and 3 from the left) showed a low but significant degree of papillation that was more comparable to the diploid controls. These transformants may therefore have been 2n prior to mating, while the others may have been of higher genome numbers. Lack of papillation by chcl-A strains transformed with pSL6 was not due to poor growth caused by irradiation, since all strains grew well on synthetic complete medium after the identical treat- ment (lower panel).

Microscopic observation of Chc- strains after transformation to Chc+: Previously we had observed that cells of Chc- strains were larger in size than typical haploids (LEMMON and JON= 1987). Possible causes of the increased size include the clathrin defect itself, increases in ploidy or nuclear number due to the clathrin defect, and a combination of both. We have no information on the size expected for a mul- tinucleate cell, but would not be surprised if it were larger than haploid size. Some information regarding these causes was obtained by microscopic examination of Ura+ Chc+ transformant derivatives of the eight Chc- strains transformed with pSL6 shown earlier in Figure 1. A typical example is shown in Figure 2. As observed previously, chcl-A scdl cells (Figure 2A) have abnormal morphology, are much larger than Chc+ haploids (Figure 2C) and usually are larger than Chc+ diploids (Figure 2D). After transformation of the chcl-A scdl cells to Chc+ (Figure 2B), cells were still abnormally large as compared to chcl-A [pSLG] haploids (Chc+) that were derived directly from dis- section and had never been propagated in the absence of clathrin heavy chains (Figure 2C). However, cells of the transformants no longer appeared granulated and rounded in appearance, nor did they display the aggregated growth habit associated with the Chc- phenotype. Growth rates and viability (as assessed by methylene blue staining, not shown) returned to nor- mal and a vacuole typical of wild type cells was now visible. Immunoblot analysis (LEMMON, LEMMON and JONES 1988) also confirmed that the Chc- cells did not produce heavy chains while the transformants did. Therefore the increased size of chcl-A scdl cells ex- amined in this study was irreversible and appeared to

be, at least i n part. associated wi th the occurrence of increased nuclear number or ploidy.

Examination of the growth heterogeneity of Chc- strains: ' I 'ht. genetic instability of chcl -A scdl cells, ;IS reported earlier (IXMMON and &NFS I Wi), was also noticed i n their growth properties. Growth rates of spore clones often incre;lsed wi th time i n liquid cul- ture, Chc- cells gave rise to faster growing papillae on plates. and small colony isolates gave both I;lrge and S I I I ~ I I I colonies upon restreaking. To es;~minc this fur- ther a more estensive analysis of spore clone 0.J3 l 19- 240 was carried out. The original spore clone was streaked for single colonies and large and small colony isolates were esamined microscopically after growth in liquid medium. Small colony isolates grew i n larger aggregates and had many more de;d or lysing cells t h;~n the faster growing isolates. The fraction of' dead cells, detected b v methylene blue staining, w a s 8- 1096, I-% and less than 0.1% for slow growing Chc-, fast growing Chc- and wild-type cells, respectively. Also, the average cell size of slow growing strains ;tppeared larger than that of fast growing Chc- strains; however, even the better growing isolates were at least ;IS large ;IS Chc' diploids (not showl). T o assess the genome content of these large i111d

small colony isolates, crosses to B.13 1 19-3.51) were

nlacle and tetrads were dissected fronl t w o zygotes for tach cross. Crosses to small colony isolates of 2413 yielded 4n (or . in) segregation, while better growing isolatcs yielded inviable spores, the result WC w o u l d espect for triploid segregation. This indicates t h a t the genome copy number of' the slow growing isolates MXS

greater th;ln the faster grol\ing popul;~tion. The cell size observed from microscopy is consistent w i t h the inferred genome numbers o f ' the two populations. -fherefore, these studies suggest that increasing the number of genomes of the cell does not neccss;arily correlate w i t h the generation of better growing chcl- A derivatives.

Evidence that faster growing Chc- cells may result from selection for suppressor mutations that further compensate for lack of clathrin: "I'olyploitly" in the rhcI-A::I"J'2 scd I strains derived from B.J:3068, the original diploid transforrnation recipient for the first series of studies, occurred soon after spore germina- tion ;Ind out-growth. Faster growing papillae were observed in the first master patches and "ploidy" changes were observed immediately in crosses. I n order to examine whether the early appearance of "polyploids" was conlnlon among a l l rhcl -A st rains. a second set of diploid transformation recipients (I3J34.50 and DJ3449) \vas made (see Table I for construction) and gene repl;lcement was performed, generating BJ3539 (Ado) and I3J3.343 (A39) respec- tively.

Upon streaking for single colonies, rhrl-A3Y-bear- ing spore clones gave large and small colonies. How- ever, when esamined in crosses only 2n segregation was obtained (Table 20). As ;I consequence, a large scale population screen was undertaken using a C a n r

test for ploidy. Patches of cells from tetrads from BJ3.543 and 13J3.539 were passaged two more times and then mass matings were performed between these C A S I " spore clones and haploid can]' strains. Proto- trophs resulting from matings were tested for their ability to papillate to Can' by mitotic recomhination on canavanine plates (Table 3). Since CAIYI' is domi- nant to ranl', mating of a haploid Can' to the Canr strain would result i n extensive papillation to Can', but nlating of Can'cells of higher nuclear number or ploidy to the Can' strain would not result in significant papillation.

Among the rhr l -A strains from I3J3.543 and 0.J3.539, four out of seven showed extensive papillation of all prototrophs from the matings to a haploid Can' strain, indicating that the Chc- cells in these populations were still haploid. However. both derivatives from eJ3.539 showed a significant nurnher of nonp~pillating proto- trophs after mating, and one of five Chc- strains csamined from I3J3.543 showed a pattern indicating cells of higher ploidy or nuclear number in the pop- ulation. I 3 v contrast, every chcl-A derivative of

34 S. K. Lernrnon et al.

TABLE 3

Can' test for genome copy number by crosses of Can' spore clones to Chc' Can' strains

Can8 Strain" Papihing Nonpapillating numbersb Inferred genome

A. Mass nlatings 1. Chc- 3543-1A 32 0 n

- l D 1 28 n,22n -2C 32 0 n -5B 29 0 n -5c 32 0 n

2. Chc- 3539-18 0 32 -2c 20 3

2 2 n n ,22n

3 . Chc- 31 19-248 1 32 -24D

n ,22n 0 31

-29C 2 2 n

0 26 22n -34D 0 32 22n

4. Chc- 3331-3D 0 32 22n 5. Chc+ 3543-1B 30 0 n

-2A 31 1 -2D 31 0 n -6C 32 0 n

n ,22n

B. Crosses to large and small colony-forming derivatives of Chc- strains'

1. 3539-1B Large 0 20 2212 Small 0 20 22n

2. 3543-1D Large 0 10 2 2 n Small 7 2 n ,22n

3. 3543-5B Large 19 I d n,22n Small 20 0 n

a The Cans strains derived from tetrad analysis of 853543 or BJ3539 were mass mated to a Chc+ Can' strain of the opposite mating type (BJ4203 or BJ4204). Cans strains from BJ3119 and BJ3331 were mass mated to Chc+ Can' strains BJ2649 or BJ2651. Papillation to Can' was tested on prototrophs as described in MA- 'TERIALS AND METHODS. Papillating indicates that an individual prototroph showed extensive formation of papillae canavanine plates, while nonpapillating prototrophs showed little.

'The inferred genome numbers of a Cans strain was n if the prototroph gave papillating colonies, 2 2 n if the prototroph gave nonpapillating colonies.

' Chc- strains were streaked for single colonies, four large and four small colonies were crossed to Can' testers and prototrophic colonies were selected. Up to five single prototrophic colonies from each individual cross were then tested for formation of papillae on canavanine medium.

In most cases each large or small Chc- colony crossed gave prototrophs with the same papillation pattern. However, the cross of one large colony derived from BJ3543-5B gave four zygotes that formed papillae on canavanine and one that did not.

BJ3068 yielded few or no papillating prototrophs after mating, while Chc+ spores derived from all of the diploid parents rarely yielded a nonpapillating prototroph. A sampling of data from such a survey is shown in Table 3.

The Canr test was also used to examine fast and slow growing Chc- derivatives of spore clones from BJ3539 and BJ3543 (Table 3B). When BJ3539-1B large and small colony isolates were crossed to the Can' tester, zygotes gave only nonpapillating patches. For BJ3543-1D, the crosses of large colony Chc- isolates gave results indicating all were of greater than In genomes, while zygotes examined from crosses of small colony isolates indicated that this Chc- popula-

TABLE 4

Linkage data for CHCl and KEXI

Tetrad type

Cross Loci pairs P N T cM"

A. BJ3444 X BJ3398' CHCI-KEXI 29 0 1 1.7

ADE5-KEX1 22 0 8 13.3

B. BJ3444 X BJ3491 CHCI-KEX1 48 0 2' 2.0

CHCI-ADE5 22 0 10 15.6

CHCI-ADE5 33 0 16 16.3 ADE5-KEXI 31 0 18 18.4

( 1 949). Map distances (cM) were calculated by the method of PERKINS

' Due to spore inviability the analysis of BJ3444 X BJ3398 was based on 21, 12, and 1 tetrads with 4, 3 and 2 viable spores respectively. Where possible, the phenotypes of the inviable spores were inferred. In two tetrads [KIL-kl] was lost and these were not analyzed for k e x l .

These tetrads had three viable spores; the phenotype of the fourth spore in each was inferred.

tion was mixed (In and 2 2 n ) . Almost all small and large colony isolates from 3543-5B generated 2n zygotes in crosses to the Canr strain, and therefore both large and small colony derivatives were largely In. Since Chc- strains grow poorly on YEPG, a tet- razolium overlay assay (OGUR, ST. JOHN and NAGAI 1957) was used to test for respiratory deficiency and in no case was the difference in growth phenotype of fast and slow growing Chc- populations attributable to the absence and presence, respectively, of petites.

Our conclusion is that, although chcl -A strains show increases in their genome content at high frequency, the genetic background can influence the rate at which this occurs or the growth advantage that poly- ploid and/or multinucleate strains enjoy. In addition, the fact that some Chc- spore clones generated better growing derivatives without a commensurate increase in genome number indicates that faster growing Chc- cells can also result from selection for suppressor mutations that further compensate for the lack of clathrin.

Mapping of CHCl: CHCl was localized to a band that contained chromosomes VII and XV by hybridi- zation of a 1 .8-kb 32P-labeled Hind111 fragment from the 3' end of CHCl to a blot of yeast chromosomes separated by OFAGE (not shown). Further crosses to a set of mapping strains (GABER et al. 1983) confirmed the location of CHCl on the left arm of chromosme VU, linked to ADE5 (1 7.5 cM).

T o map CHCl more precisely, three point crosses involving ADE5, CHCl and K E X I , which had been mapped approximately 24 cM proximal to ADES (WICKNER and LEIBOWITZ 1976), were set up. Since Chc- strains grow poorly, strain BJ3444 was con- structed, in which the LEU2 disruption-deletion ver- sion of CHCl was integrated adjacent to CHCI, such that CHCl is marked with LEU2 but is not itself disrupted. Two crosses were made (BJ3444 X BJ3398

Clathrin-Deficient Yeast 35

CHC I """""_ + mRNA

C B\HqG R P YH H H HRHIR H R R P C B

2 - PROBE

PH HPR B C,H F I H C B k T G R P G R P C -

KEXI REGION H 1 kb

and BJ3444 X BJ3491) and tetrad analysis was per- formed (Table 4). Close linkage of KEXl and CHCl was indicated, since no parental ditypes and only three tetratypes between the two loci were obtained from analysis of 80 tetrads.

Other evidence for the close linkage of CHCl and KEXl was obtained from comparison of pSL6 and pAD17, which contains the KEXl region. The restric- tion maps of the inserts overlap (Figure 3), with only 3-4 kb between CHCl and K E X l . DNA blotting ex- periments confirmed the overlap of the cloned se- quences (not shown).

Since tetrad analysis left some ambiguity as to the orientation of CHCl and KEXl relative to the cen- tromere (see Table 4), we took advantage of the genetic properties of integrated 2p plasmid DNA in yeast chromosomes (FALCO et al. 1982; FALCO and BOTSTEIN 1983) to order the genes. In cir+ diploids, one observes instability of the chromosome into which 2p DNA is inserted, apparently due to chromosomal breaks that follow integration of endogenous episomal 2p circle by recombination. This results in loss of all markers distal to the site of integration, and can result in loss of proximal markers and even the entire chro- mosome. Broken chromosomes are often repaired using information from the homologous chromosome.

A YEp24 plasmid containing a portion of the 5' region of CHCl (pSL5) was cut at the unique BstEII site in the insert sequences to direct integration be- tween KEXl and CHCl (Figure 4A). The order of the sequences on chromosome VZZ of the resultant strain (BJ3083) was KEXl , URA3,2p DNA, C H C l . Two leu2 ura3 ADE5 CHCl::2p,URA3 KEXl derivatives of BJ3083 were obtained (BJ4469 and BJ4470) and were crossed to BJ3916 (leu2 ura3 ade5 CHCl kexl- A::LEU2). Since the resultant diploids were homozy- gous for leu2 and ura3 at their normal chromosomal loci, we were able to follow recombination or repair between KEXl and CHCl on chromosome VZZ from the analysis of the Ade, Leu and Ura phenotypes of spore progeny. Therefore, diploids that had destabi- lized chromosome VZZ (Ade- Ura- Leu+ segregants) were examined by random spore analysis.

The types of events to be expected depend upon the order of CHCl and KEXl (Figure 4). If CHCl is

FIGURE 3.-CHCI and KEXl clones have overlap- ping restriction enzyme maps. Upper: CHCl insert from pSL6. Lower: insert from pAD17 that contains KEXl. The 1.9-kb BglII fragment used as a probe for DNA blot analysis to confirm the overlap of the two clones (data not shown). Restriction enzymes are: B, BamHI; C, ClaI; G , BglII; H, HindIII; P , PstI; R, EcoRI.

distal to KEXl (Figure 4B), arm breakage can result in production of two different classes of Ura- diploids, those in which the KEXl allele is lost (Figure 4B, left) and those in which it survives (Figure 4B, right). The former class of diploid yields only Leu+ Kex- Ura- Ade- progeny. The latter class of diploid gives two kinds of spore progeny: half of Leu+ Kex- Ura- Ade- phenotype and half of Leu- Kex+ Ura- Ade- pheno- type. If CHCl is proximal to KEXl (Figure 4C), arm breakage will always lead to loss of URA3, KEXl and ADE5, since all are distal to the breakage site at 2p. All spore progeny will be of phenotype Leu+ Kex- Ura- Ade-, regardless of the extent of resection of the broken arm.

Of eight Ade- Ura- diploid segregants obtained from BJ4469 X BJ3916, seven yielded only one spore type (Leu+ Kex- Ura- Ade-) (although alleles at the MAT locus and other segregating nutritional markers were present in nearly equal frequencies). One diploid yielded seven Leu+ Kex- Ura- Ade- spores and nine Leu- Kex+ Ura- Ade- spores. Upon dissection of a few tetrads from this diploid the expected 2:2 segre- gation for Leu and Kex and other segregating mark- ers was observed. Thirty Ade- Ura- diploids were examined from BJ4470 X BJ3916, and 15 gave both Leu+ Kex- Ura- Ade- and Leu- Kex+ Ura- Ade- spore types. These results are consistent with the conclusion that KEXl is 1-2 cM (and 3-4 kb) proximal to CHCl on the left arm of chromosome VZZ.

DISCUSSION

We have identified at least two sources of genetic instability in clathrin-deficient strains, the first involv- ing changes in genome copy number. Several inde- pendent tests indicate that cells with increased num- bers of genome copies per cell occur with high fre- quency in some Chc- strains. The tests involving mating or transformation may provide unreliable measures of the frequency of cells of higher ploidy or genome content, since it is possible that Chc- "poly- ploids" mate more readily than do Chc- haploids or are more receptive to transformation than Chc- hap- loids. However, by visual inspection, the proportions of large cells in the Chc- populations we examined were high, and, upon transformation to Chc+, cells

36 S. K. Lemmon et al.

A f l D N A

K f X l REGION

I CHCl ORF

3

- Vector DNA - Chromosomal DNA - Homologous Regm

B - KEXl URA3 2p CHCl ADE5

kexl : : LEU2 CHCl ade5

A Annbreakgearsdloss

KEXl " kexl: : L N 2 CHCl ade5 nkex l : :LN2 CHCl ade5

1

1 1

v

ma" Pick Ura ,Me- I

kexl : :LN2 CHCl ade5 KEXl CHCl ade5

kexl: :LEU2 CHCl ade5 kexl: :LEU2 CHCl ade5 v

" sporulaticm

spore Leu+ m- ura- Me- Leu' W- Urn- Me- (50%) Types:

Ieu- W+ Ura- Me- (50%)

C r\ CHCl 2p LIRA3 KEXl ADE5

CHCl kexl : : LEU2 ade5

AAnn breakage and loss

-0- CHCl

CHCl kexl : : LEU2 ade5 CHCl k e x l : :LEU2 ade5 I

apcrair I Pick Ura',Me' I

spore I s E O l u l 2 1 CHCl kexl : :LEU2 ade5 CHCl kexl : :LEU2 ade5

CHCl kexl : :LEU2 ade5 CHCl kexl : :=2 ade5

Types: Leu+ m- uxa- Ad@- Leu+ m- urn- "

FIGURE 4.--Integration of 2 p DNA sequences at the clathrin heavy chain locus to determine relative centromere orientation of CHCl and KEX1. A, Model for the integration of pSL5. The plasmid was linearized at the unique BstEII site to direct the integration between K E X l and CHCl. Restriction enzymes are B, BamHI; G , BglII; Bst, BstEII. B, Expectations for arm loss and repair due to 2p integration if CHCl is distal to K E X l . C, Expectations for arm loss and repair due to 2p integration if CHCl is proximal to KEXl . For B and C the circle indicates the centromere on each of the chromosome homologs. On the left are the expectations if breakage and repair from the ho- mologous chromosome occurred proximal to both CHCl and KEXl . On the right are the expectations if breakage and repair from the homologous chro- mosome occurred between K E X l and C H C l , leav- ing the proximal marker intact. Not shown are the cases where the whole chromosome would be lost. The spore expectations for those events would be the same as the events shown on the left in B and C. Note that our results are consistent with expec- tations shown in B, that is, KEXl is proximal to CHC 1.

remained large. Since increases in cell size have been correlated with increases in ploidy (MUNDKUR 1953; MORTIMER 1958) or nuclear number (CONDE and FINK 1976), the numbers of abnormally sized cells suggests that the frequency of "polyploid" cells is high in these Chc- cultures, in agreement with the other tests.

Previously we observed that Chc- cells growing logarithmically can undergo aberrant nuclear division and usually retain both progeny nuclei in the mother cell (LEMMON and JONFS 1987). In some cells, pre-

mature migration of the nucleus to the site of bud initiation appeared to have occurred. Although the percentage of such unusual divisions was low, it was difficult to determine the frequency at which they occurred, because only a small proportion of cells were in mitosis, and it is unknown how many cells were already derivatives of aberrant division. In ad- dition, the genetic tests for "ploidy" that we employed, viz., segregational analysis and frequency of mutation to canavanine resistance, do not distinguish between polyploid cells and multinucleate cells. However, if

Clathrin-Deficient Yeast 37

the rate of discoordinate nuclear division is as low as it appears, then we suggest that there must be a growth advantage for Chc- cells of higher genome copy number that enables them to take over the population as rapidly as they do. Only when we obtain conditional mutations in the heavy chain gene will it be possible to quantitate the actual frequency of ab- normal divisions and substantiate or refute the infer- ence that cells with higher genome content per cell have a growth advantage.

T o determine whether other Chc- strains gave com- parable increases in genome content, we extended our analysis to another pair of diploid transformation recipients whose haploid parents were partially de- rived from a set of strains that were thought to be congenic to ours. Some Chc- spore clones from BJ3539 and BJ3543 showed a significant frequency of “polyploids” in the population, whereas others did not. The frequency of “polyploid” clones among these Chc- strains was substantially higher than the fre- quency observed in Chc+ strains, but was significantly lower than that seen among the Chc- strains derived from BJ3068. Clearly other genetic factors influence the rate at which “polyploids” take over the popula- tion. At present we cannot distinguish between a genetic factor that alters the rate of production of “polyploids” from one that results in a difference in the growth advantage that “polyploids” may enjoy. Furthermore, there appear to be genetic backgrounds in which the frequency of increased genome content of Chc- cells is very low, since PAYNE et al. (1987) did not observe the occurrence of “polyploids” in a series of independently isolated chcl -A strains, although an extensive examination like ours was not undertaken.

The identification of zygotes as tetraploid, resulting from mating of a Chc- cell with triploid genome content to a haploid Chc+ cell, was unequivocal in cases where meiotic analysis of the progeny spores of the tetraploid zygote were also performed. That tri- ploid zygotes occurred, resulting from mating of a Chc- cell of diploid genome content with a haploid Chc+ cell, was inferred in cases where meiotic analysis gave few viable spores, but remains only a conjecture. Since even pentaploid cells yield reasonable frequen- cies of viable meiotic progeny (MORTIMER 1958), we usually could not distinguish tetraploid zygotes from zygotes of higher ploidy unless spore progeny were analyzed further. Thus, although we believe that a population of Chc- cells contains cells of one, two, three and possibly higher numbers of genomes per cell, we have unequivocal evidence only for cells with haploid or triploid genomic contents.

The tetraploid zygotes contained three copies of the genome of the Chc- cell and one copy of the genome from the Chc+ cell. Endomitosis by itself cannot give a triploid cell. Various ad hoc explanations

can account for three genome copies per cell. For example, an aberrant nuclear division yielding a bin- ucleate cell with two haploid nuclei could be followed by a second asynchronous and aberrant nuclear divi- sion of one nucleus (LEMMON andJoNEs 1987) to give three haploid nuclei (not seen) or one haploid and one diploid nucleus. T o generate the tetraploid zygote nucleus, one must postulate multinuclear fusion of nuclei in the zygote or fusion of the nuclei within the Chc- cell. We have no evidence to distinguish among the possibilities, but we are left with the impression that more than one kind of abnormal nuclear behavior occurs.

We often observed that slow growing Chc- strains gave rise to faster growing strains but never observed the opposite change. Frequently the increased growth rates of the variants occurred without an increase in “ploidy” of the strains (see Table 3B especially). These findings suggest that there is a second cause of genetic instability of Chc- strains and that there is a strong selection for suppressor mutations that compensate for the lack of clathrin. Further studies will be re- quired to identify these other genes.

Mutations in several other genes result in cells with increased ploidy, for example, cdc31 (SCHILD, ANAN- THASWAMY and MORTIMER 198 l), n c d l (THOMAS and BOTSTEIN 1986), karl (ROSE and FINK 1987) and espl (BAUM et al. 1988). However, each of these appear to have defects in spindle pole duplication and/or chro- mosome separation. Since, chcl-A cells grown at 30” have nuclei and spindle poles that appear normal and we saw no evidence for aneuploidy, we infer that Chc- cells have normal spindle pole duplication and chro- mosome separation.

We have mapped CHCl 1-2 cM from KEXI on the left arm of chromosome VZI. The location is consistent with the results of PAYNE et al. (1987) who mapped CHCl between ADE5 and LYS5. By using an integrant that contained 2p DNA between CHCl and KEXI, we unambiguously determined the order to be centrom- ere-KEX1-CHCI. This 2~ mapping procedure should be generally applicable to the ordering of any two genes that are far from the centromere. We note that other chromosome breakage procedures, such as the technique of physical mapping of DNA by chromo- some fragmentation (VOLLRATH et al. 1988) could also be adapted for ordering closely linked loci.

We thank CARL FALCO, PHILIP HIETER, H. BUSSEY and A . COOPER for materials, strains and plasmids; MICHELLE MORITZ for help in generating the 2 p integrant; CLARENCE CHAN for the suggestion of mating to Can‘ haploids to determine ploidy: and ROBERT K. MORTIMER and rnembrrs of our laboratory f o l - their helpful discussions.

This work was supported by I1.S. Public Health Service grants GM29713 and AM 18090 to E.W.J. and A106884 and GMJ699.5 to S.K.L.

38 S. K. Lemmon et al.

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Communicating editor: M. CARLSON