Rescue of a single yeast artificial chromosome from a cotransformation event utilizing segregation at meiosis

123 GATA 10(6): 123-127, 1993

ORIGINAL ARTICLES

Rescue of a Single Yeast Artificial Chromosome from a Cotransformation Event Utilizing Segregation at Meiosis ANDREA H. NI~METH, NEIL HUNTER, MICHAEL P. COLEMAN, RHONA H. BORTS, EDWARD J. LOUIS, and KAY E. DAVIES During the construction of yeast artificial chromosome (YAC) libraries to facilitate mapping of the human genome, two YACs may be cotransformed into the same yeast cell, making further analysis very difficult. We present a simple method to rescue the required YAC that utilizes the segregation of chromosomes at meiosis. In brief, we crossed the cotransformed yeast cell with a non-YAC-containing strain and induced the resulting diploid to sporulate and undergo meiosis. The new haploid generation included some yeast cells that contained only the desired YAC. These YACs were analyzed by conventional methods. To exclude the possibility that major rearrangement occurred during the procedure, we analyzed the YACs with restriction enzymes that cut only rarely. We conclude that this is a useful technique to rescue cotransformed YACs.

entire human genome. During the construction of YAC libraries, more than one YAC clone may be cotransformed into the yeast Saccharomyces cerevisiae, making further analysis of the desired YAC very difficult. We describe here a method to "rescue" the desired YAC by using segregation at meiosis. Saccharomyces cerevisiae has two different mating types: MATa and MATot. Conjugation occurs when strains of opposite mating type are mixed. We therefore crossed the host strain AB1380 (MATa) containing both YACs with a suitable non-YAC-containing strain, EJL538-1A (MATot). The resulting diploid cell contains 2n yeast chromosomes and two YACs. Meiosis and sporulation of the diploid cells is induced by nitrogen starvation to form haploid nuclei. Each meiosis results in four nuclei, known together with their cytoplasm as ascospores. The four spores are packaged as tetrads surrounded by a thick-walled sac, the ascus. The spores can be dissected apart and each of the haploid progeny grown separately. If normal mitosis of the diploids and correct distributive segregation of the YACs during meiosis occurs, then each new haploid cell will contain In yeast chromosomes and one YAC [1] (Figures 1 and 2). In fact, one or other YAC may be duplicated mitotically, and aberrant segregation of YACs at meiosis may also occur. The resulting progeny may therefore contain only one YAC (as desired in this situation), both original YACs, or lose the YACs completely. The haploid meiotic progeny were therefore screened by growing on selective media to identify those containing YACs and then analyzed by pulsed-field gel electrophoresis (PFGE) to identify which contained the single YAC of choice.

Introduction One of the crucial new technologies associated with mapping the human genome has been the stable propagation of large DNA segments as yeast artificial chromosomes (YACs). YACs can be assembled into overlapping "contigs" that span the regions of interest and should eventually cover the

From the Molecular Genetics and Yeast Genetics Groups, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, England. Address correspondence to Dr. A.H. N~meth, Molecular Genetics Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU England. Received 3 November 1993; revised and accepted 3 December 1993.

Materials and Methods A 4X YAC library from the Imperial Cancer Research Fund [2] was screened by hybridization with the X-specific probe 2bC6, at locus DXS226. Chromosome plugs were prepared by conventional methods [3] and analyzed by PFGE. Hybridization with the 1.6-kb BamHVPvuII fragment of pBR322 specific for the "right arm" of pYAC4, revealed a cotransformation event, with two YACs of 630 kb and 500 kb in one yeast cell. Hybridization with the original DXS226 p r o b e showed that the smaller of the two YACs was the desired one. To rescue this YAC, the following strategy was used.

© 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010 1050-3862.793/$6.00

124 GATA 10(6): 123-127, 1993

A.H. N6meth et al.

X

Haploid (1 n)

~b'/ Non YAC Yeast Strain YACs

Diploid (2n) Inteq~hase Duplication

V

Meiosis I

x"

Figure 1. Normal distributive segregation o f YACs at meiosis. Two YACs (represented by long and short bars, r e s p e c t i v e l y ) are initially c o n t a i n e d within a single haploid cell. The cell is mated with a non-YAC haploid cell to produce a diploid. D N A is duplicated during interphase, and the subsequent meiosis results in four new u r a + haploid cells, two containing each YAC.

\

Meiosis II

t

t

t

t

(i) (i) I

All 4 ura+

New generation

Haploid (ln)

I

(1) The YAC-Containing Strain Was Crossed with a Suitable Non-YAC Host YACs are propagated in host strain AB1380 that contain the following genetic markers: ura3-52, trpl, ade2-1, canl-lO0, lys2-1, and his5 [2]. The non-YAC strain EJL538-1A was chosen because it contains the following genetic markers: ura3-52, trp-H3, ade2-101, cyh2-R, and his4-R. (The strain EJL538-1A will be available to researchers on request). Any other appropriate combination of mating type MATa and MATs with complementary markers would be suitable.

(2) Mating Strains to Form Diploids The YAC host strain was grown as a 3 x 0.5-cm " p a t c h " on a YPD plate (Bacto-yeast extract, 1%; Bacto-peptone, 2%; glucose, 1%; and Bacto-agar, 2%) [4]. The non-YAC strain was streaked onto the YAC strain by using a sterile toothpick and mixed on the plate. The plate was incubated overnight at 30°C.

(3) To Select for Diploids The mated cells were streaked out on minimum media plates plus adenine [4]. The diploid yeast

© 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

125 GATA 10(6): 123-127, 1993

Rescue of a Cotransformed YAC by Meiosis

The strains used are sensitive to excessive glusulase treatment. Excessive exposure to glusulase leaves only single spores rather than dissectable tetrads. The time in Glusulase is critical: some strains require up to 10-15 min in stock glusulase before water is added to give dissectable tetrads. The tetrads were dissected with a micromanipulator (Singer Instruments) onto YPD plates. (An excellent account of tetrad dissection is contained in Current Protocols in Molecular Biology [5].) The individual spores were grown at 30°C for 3 days. The colonies were replicated using velvet onto synthetic complete minus uracil (sc-ura) and synthetic complete minus tryptophan (sc-trp) [4] to screen for the YAC-containing haploids. The colonies were also replicated onto fresh YPD master plates. Because of unusual mitotic and meiotic events, several tetrads should be analyzed to ensure that at least one of the haploid colonies contains the desired YAC (see the Discussion).

A

L

J

Ura-

B

ura+

0

I

I

ura-

ura+

C

I

I

I

I

ura-

ura+

°

(6) To Analyze Segregants t ura-

J ura+

Figure 2. Mechanisms resulting in unusual haploid progeny.

onL"~"ffffd short bars represent the two different cotransformed YACs: (A) Normal segregation with chromosome loss. (B) Mitotic endoduplication with chromosome loss. (C) Nondisjunction at meiosis I. (D) Nondisjunction at meiosis II.

cells were grown for 3 days at 30°C. Only mated diploids with complementing markers grow~

(4) Inducing Sporulation of Diploids Ten diploid colonies were then picked and grown as 4-cm 2 "patches" on master YPD plates overnight at 30°C. The colonies were printed onto a sterile velveteen square and replicated onto potassium acetate plates [4]. Sporulation was left to proceed at 250C on the bench for 3 days.

(5) Preparation of Tetrads for Dissection Five tetrads from each of five diploids were selected for dissection. Approximately half of each patch was picked into 50 i~1 Glusulase N E N Dupont (Stock, 1:10 dilution), and 0.5 ml distilled water immediately added. Glusulase is derived from the intestinal tract of snails and contains multiple enzymes that break down the tough ascus surrounding the spores. The spores were left for 1 h at room temperature.

The master plates were compared with sc-ura and sc-trp selective plates to reveal the ratio of nonYAC-containing to YAC-containing haploids. Assuming correct distributive segregation during meiosis [1], it is expected that the patterns will be 0 u r a - : 4 ura+ colonies (see Figure I). Tetrads giving a 0 u r a - :4 ura+ or 1 u r a - :3 ura+ pattern are likely to contain colonies with single YACs (see Figure 2). Those with a parental 2 + :2 - ratio are likely to still contain haploids with both YACs. Out of 25 tetrads, seven were suitable for further analysis. Those that did not have four viable spores and those with a 2:2 segregation pattern were not analyzed. A total of 22 haploid segregants from the seven tetrads were selected as potential carriers of the desired YAC. These segregants were grown overnight in YPD broth at 30°C and glycerol stocks and chromosome plugs prepared. The plugs were analyzed by PFGE (LKB pulsaphor, 175 V, 1-min switching time, 24-h run in 0.045 M Tris-borate and 0.001 M EDTA, pH 8.0). The desired strain was identified by staining the gel with ethidium bromide. This was confirmed by conventional Southern analysis and hybridization with the 1.6-kb BamHI/PvulI fragment of pBR322 specific for the right arm of the pYAC4 vector and then the DXS226 probe originally used to screen the YAC library.

© 1993 Elsevier Science Pubfishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

126 A.H. N6meth et al.

GATA 10(6): 123-127, 1993

T a b l e 1. R e s u l t s o f T e t r a d s A n a l y z e d

Tetrads analyzed

Tetrads without 4 viable spores

Tetrads with 2 u r a - :2 ura+ segregation ratio

Tetrads with 1 u r a - :3 ura+ segregation ratio

25

8

10

6

Tetradl Segregants

1

Tetrad 2

2

3

4

5

Tetrad 3 6

7

8

Tetrad 4 9

10

11

12

Figure 3. The 22 YACs prepared by stanT~d---m--~thods after meiosis. LKB Pulsaphor, 175 V, l-min switching time, 24-h run. Hybridization probe is right arm of pYAC4 vector. Note that lanes 1, 3, 7, and 9 show segregants still containing two YACs; lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, and 19 show segregants containing the smaller (desired) YAC; lanes 5, 11, 13, 15, 17, 20, and 21 show segregants containing the larger YAC; and lane 22 shows two signals of smaller size suggesting an unusual rearrangement.

630kb500kbTetrad 5 Segregants

500kb -

14

13

~

15

Tetrad 6 16

17

18

Tetrsd 7 19

20

Tetrads with 0 u r a - :4 u r a + segregation ratio

21

22

~

m

To confirm that there had been no internal rearrangements that had left the size of the whole YAC unchanged, we analyzed two of the 500-kb (desired) YACs by partial digestion with the restriction enzymes BssH II, Eagl, and Sac II, which cut rarely in human-derived DNA. The YAC DNA plugs in agarose were digested for 45 min with a range of enzyme concentrations between 0.25 U/ixl and 0.0004 U/~I. Half of each plug was stored in 0.5 M EDTA while the remainder were used to assess the extent of digestion by PFGE. The stored plugs yielding the most representative mixture of partial digestion products were reanalyzed by PFGE, using conditions to separate the fragments (175 V, 20-s switching time, for 15 h), and then by Southern analysis and hybridization with the 1.6-kb right YAC arm probe and then the 2.5-kb BamHI/PvulI fragment of pBR322 specific for the left arm of the YAC (Figure 4). This technique was used to construct a restriction map of -10-kb resolution.

Results o f the 25 tetrads dissected, seven were suitable for further analysis (see Table 1). In total, 22 segregants were analyzed: ten contained the (500 kb)

BssH II Eagl Sacll 14 16 14 16 14 16 kb 500 -

400-

300

-

200 -

100

-

Figure 4. Autoradiograph showing Southern analysis of pu~ld gel electrophoresis (PFGE) of partial rare-cutter digestion products of segregants 14 and 16. The identical pattern suggests that there are no small rearrangements undetected by PFGE of undigested YAC.

© 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010

127 Rescue of a Cotransformed YAC by Meiosis

GATA 10(6): 123-127, 1993

Table 2. Results of Segregants Analyzed Tetrads with 1 u r a - : 3 ura+ segregation ratio

Tetrads with 0 u r a - : 4 ura + segregation ratio

(n = 6)

(n = 1)

Total

18 8 5

4 2 2

22 10 7

Total number of segregants Segregants with smaller YAC (500 kb) Segregants with larger YAC (630 kb) Segregants showing nondisjunction or mitotic duplication Rearrangements

m

desired YAC, seven contained the (630 kb) unwanted YAC, four contained both, and one showed clear evidence of unusual rearrangement with two fragments smaller than either original YAC (Figure 3, lane 22; and Table 2). Partial digestion of two of the rescued YACs (segregants 14 and 16) revealed identical restriction maps indicating that no rearrangement or recombination involving a region of >10 kb had occurred (Figure 4).

Discussion A simple protocol for the rescue of a cotransformed YAC has been described. We believe that it may be extremely useful to investigators using YACs particularly for the human genome-mapping project. Potential sources of complication with this method include mitotic endoduplication, premeiotic and meiotic chromosome loss, aberrant segregation, and rearrangements or loss of the chromosomes. We found nearly all of these events during this experiment, but because we analyzed a sufficient number of segregants, we were still able to rescue the required YAC. Of the seven tetrads analyzed, only one (segregants 13-16) displayed normal 0 u r a - :4 ura + segregation. The other six all showed a l u r a - : 3 ura+ ratio. In two tetrads (segregants 1-3 and 7-9), there are five YACs in total, suggesting an endoduplication event in mitosis with subsequent nondisjunction and chromosome loss at meiosis (Figure 2). The remaining four tetrads showed either nondisjunction (at meiosis II) or chromosome loss. There are several explanations for the events that we observed. Chromosomes are thought to separate at meiosis by at least two different mechanisms. In one, recombination events and the formation of chiasmata are thought to ensure normal separation [6]. Nonhomologous YACs may use another mechanism, known as distributive disjunction, which does not require recombination, but is not able to ensure disjunction 100% of the time [1]. Intrachromosomal recombination between Alu or other

repeat sequences might have been a complication as yeast chromosomes recombine at meiosis more frequently on a per base pair basis than human chromosomes, and homologous YACs recombine at a frequency comparable to native yeast chromosomes [6]. We observed only one gross rearrangement, however, and we excluded smaller rearrangements by analyzing the segregants by restriction digests with rare-cutter enzymes. An alternative to dissecting tetrads by using micromanipulation is to isolate random spores by overdigestion with Glusulase prior to plating for single colonies. A description of random spore analysis can be found in Sherman et al. (1986) [4]. However, more colonies will have to be screened (in the region of 40--50) than from tetrad analysis, as preselection for the appropriate type of segregation is not possible. We therefore hope that the method described here will be useful to investigators who encounter cotransformation events when working with YACs. We thank Dr. Mark Hirst and Dr. Samantha Knight for helpful comments and the Imperial Cancer Research Fund and Dr. Giinther Zehetner for providing the YAC library. This work was supported by the Medical Research Council (MRC), the Human Genome Mapping Project, the British Retinitis Pigmentosa Society, and the WeUcome Trust. A.H.N. is an MRC Training Fellow.

References 1. Dawson DS, Murray AW, Szostak JW: Science 234:713717, 1986 2. Lehrach H, Drmanac R, Hoheisel J, Latin Z, Lennon G, Monaco AP, Nizetic D, Zehetner G, Poustka A: In Davies KE, Tilghman SM (eds): Genome Analysis, vol 1. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1990, pp 39--81 3. Bickmore W: In Anand R (ed) Techniques for the Analysis of Complex Genomes. New York, Academic, 1990, pp 19--38 4. Sherman F, Fink GR, Hicks JB: Methods in Yeast Genetics. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1986 5. Treco D, Winston F: In Ausubel FM, et al. (eds): Current Protocols in Molecular Biology, vol 2. New York, John Wiley and Sons, 1993, Unit 13.2.4 6. Sears DD, Hegemann JH, Hieter P: Proc Natl Acad Sci USA 89:5296--5300, 1992

© 1993 Elsevier Science Publishing Co., Inc., 655 Avenue of the Americas, New York, NY 10010