Assembly and extension of yeast artificial chromosomes to build up a large locus

GENE AN INTERNATIONAL JOURNAL C~ENE5 AND OENOME,5

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Gene 177 (1996) 195-201

Assembly and extension of yeast artificial chromosomes to build up a large locus Andrei V. Popov a, Christoph Bt~tzler a, Jean-Pol Frippiat Marianne Brtiggemann a,,

b,c,

Marie-Paule Lefranc c,

a Department of Development and Genetics, The Babraham Institute, Babraham, Cambridge CB2 4AT, UK b M R C Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, UK c Laboratoire d'lmmunoGbnktique Molkculaire, lnstitut de G~n~tique Mol~culaire, UMR 9942 CNRS, 1919 Route de Mende, BP 5051, 34033 Montpellier Cedex 1, France

Received 29 November 1995; accepted 12 February 1996

Abstract

For the assembly of a large human locus, overlapping regions on yeast artificial chromosomes (YACs) and cosmids were linked up using their regions of homology. By site-specific recombination a YAC of 410 kb was created accommodating the major part of the human 2 light chain locus in authentic configuration with 28 variable (V) genes, all joining (J) segments, all constant (C) genes and the downstream enhancer. A contiguous region was first constructed from three overlapping cosmids. Each of these was linearized at unique sites in the vectors and YAC arms were ligated to the 5' and 3' ends. After cells of Saccharomyces cerevisiae were transformed with the three cosmids, YACs of 120 kb were obtained which contained the reassembled 3' J-C region in authentic configuration. The assembled YAC was further extended by mitotic recombination with a YAC containing a 280-kb region of the C-proximal part of the V gene cluster with a 15-kb 3' overlap. For this, a simple three-way selection procedure was developed involving the integration of different selectable marker genes at specific sites in the left and right YAC arms. Rare recombination events between two overlapping YACs could be identified in yeast clones able to grow in lysine- and adenine-deficient medium in the presence of 5-fluoro-orotic acid which is toxic for yeast cells containing a YAC with a functional URA3 gene. This approach made it possible to assemble and extend large YACs from an unlimited number of smaller overlapping YACs by positivenegative selection. Keywords: Saccharomyces cerevisiae; Homologous recombination; Selection vector; Site-specific integration; Transformation; Mitotic recombination; Overlapping cosmids; K A R l mutation

1. Introduction The study of large regions and gene loci has been greatly facilitated by the use of YACs for cloning, * Corresponding author. Tel. +44 1223 832312; Fax +44 1223 836481; e-mail: [email protected] Abbreviations: aa, amino acid(s); Ade, adenine; ADE2, gene encoding phosphoribosylamino-imidazole-carboxylase; amp, bacterial gene conferring ampicillin resistance; ARS, autonomously replicating element; bp, base pair(s); C, constant gene; CEN, centromere; CYH, cycloheximide; E, enhancer ; 5-FOA, 5-fluoro-orotic acid; His, histidine; lg, immunoglobulin; J, joining segment; kb, kilobase(s) or 1000 bp; L, light chain; Lys, lysine; LYS2, gene encoding c~-aminoadipate reductase; MAT, mating type; Mb, mega base pair(s); NEO, aminoglycoside phosphotransferase gene to allow selection in G418; PFGE, pulsed-field gel electrophoresis; SD, synthetic dextrose medium; TEL, telomere; Trp, tryptophan; TRP1, gene encoding N-(5'-phosphoribosyl)-anthranilate isomerase; Ura, uracil; URA3, gene encoding orotidine-5'-phosphate decarboxylase; V, variable gene; YAC, yeast artificial chromosome; YNB, yeast nitrogen base. 0378-1119/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved P H S0378-1119(96)00301-0

m a p p i n g and expression analysis (Burke et al., 1987; Nelson and Brownstein, 1994). YACs can be larger than 1 M b but more usual is a size of several h u n d r e d kb. This means that large gene loci are rarely a c c o m m o d a t e d on one YAC but instead must be m a p p e d to several overlapping YAC clones or an assembly of a large n u m b e r of cosmids. The h u m a n i m m u n o g l o b u l i n 2 light chain locus (IgL) on c h r o m o s o m e 22q11.2 is such an example. A 1.1-Mb contig, c o m p o s e d of 129 cosmid and eight YAC clones, contains 52 V2 genes, interspersed with pseudo Vs, and seven C2 genes preceded by one J2 segment each (Frippiat et al., 1995). The main attraction of using yeast as a cloning host is that its c h r o m o s o m e s including the YAC can be modified efficiently by site-specific integration (Davies et al., 1992; Davies and Brt~ggemann, 1993; Spencer et al., 1993; Yasui and Kurosawa, 1993). This allows the addition or deletion at h o m o l o g o u s sequences and the recombination of overlapping YACs by contig extension

196

A. ld Popov et al./Gene 177 (1996) 195-201

to build up a locus. Besides the generation of conventional YAC libraries (Burke et al., 1987), the high efficiency of homologous recombination of exogenous DNA transfected into yeast cells led to the development of alternative approaches for YAC construction. Recombinational cloning has been achieved by co-transfection of YAC arms, containing sequences homologous to the 5' and 3' genomic region of adenovirus type 2, with viral DNA to integrate the complete genome (Ketner et al., 1994). A similar approach exploited transformation-associated recombination by co-transfection of YAC arms with integrated Alu-repeat sequences and human DNA (Larionov et al., 1996). Furthermore, larger YACs were obtained by mitotic recombination of overlapping YACs with and without selection (Rotomondo and Carle, 1994; Den Dunnen et al., 1992 and refs. therein). In these experiments the use of yeast crossing, transfection or protoplast fusion technologies to achieve rare mitotic recombination events did establish Mb size regions; however, instability and authenticity of the resulting YACs remained a problem (Heikoop et al., 1994). Other efforts to recombine overlapping YACs relied on the use of meiotic recombination between two or more YACs assembled in the nucleus of a diploid host after mating (Green and Olson, 1990; Silverman et al., 1990; Den Dunnen et al., 1992; Sears et al., 1992; Marchuk et al., 1992; Rotomondo and Carle, 1994). Although meiotic recombination occurs very frequently in Saccharomyces cerevisiae, its use is limited not only by the rather time consuming tetrade dissection but also by a high frequency of undesired recombination events promoted by sporulation (Sears et al., 1994; Heikoop et al., 1994). To overcome this problem UV irradiation has been used in order to stimulate mitotic recombination events between two YACs in isosexual diploid hosts produced by yeast-yeast fusion (Ragoussis et al., 1992). A concern using this approach is, however, the risk of disabling the cloned region by the introduction of unwanted mutations. In the experiments presented, we used site-specific integration of selectable marker genes into opposite YAC vector arms to apply positive and negative selection pressure in order to screen for mitotic recombination events. The desired extended YAC, which contains half of the human IgL locus, was obtained from overlapping cosmids and YACs obtained in haploid yeast cells which had also discarded the unwanted, usually smaller, recombination product. 2. Results and discussion

2.1. Strategy of YAC contig construction A map of the human lg 2 light chain locus (IgL) containing V, J and C genes has been established (Frippiat et al., 1995). The analysis was carried out by

identifying overlapping YAC clones and a large number of cosmids. A joint region essential for expression analysis containing V, J, C and putative control regions, such as the 3' lg 2 enhancer, was not contained in a single YAC or cosmid clone. Therefore we linked the V gene containing YAC 35B2 (Frippiat et al., 1995), termed V2 YAC in our experiments, with three overlapping cosmids, N97B2, N59D10 and N78H4 (Frippiat et al., 1995), to produce a locus containing half the number of V genes and the whole J-C region. In Fig. 1A the large region of the human 2 light chain locus represented by the V)o YAC and the three C2 cosmids is illustrated. The strategy to construct a 410-kb YAC covering a genomic region of about 380 kb focussed first on the assembly of a 120-kb region from the three overlapping cosmids (see Fig. 1B). Reconstitution of the C2 region on a YAC and subsequently linking this YAC via the 15-kb overlapping region with the original 280-kb V2 YAC, extended to about 300 kb by the addition of a selectable marker gene, would provide a contig with 28 V genes and seven J-C genes, including the enhancer downstream region.

2.2. YAC construction from overlapping cosmids As illustrated in Fig. 1B, the three cosmids used for the YAC assembly contained overlapping homologous regions of 15 and 6 kb. Further analysis of restriction digests identified unique sites in the vector sequence. BssHII was used for the linearization of cosmid N78H4, whilst EagI released the inserts from the vector sequences in cosmids N97B2 and N59D10. To accommodate the YAC vector arms the most 5' cosmid, N97B2, was ligated with the centric or left arm of pYAC-RC (Marchuk and Collins, 1988), and the most 3' cosmid, N78H4, was ligated with the right or acentric arm of this vector which provides the URA3 gene for selection in uracildeficient medium. For YAC construction, DNA from the left and right YAC arm cosmids was mixed with DNA from the overlapping cosmid N59D10, which links up both regions, at a ratio of 1:1:3. Co-transformation of yeast strain AB1380 according to Burgers and Percival (1987) (see legend to Fig. 1 for genotypes of yeast strains) resulted in about 2000 colonies, from which 20 were analysed for the presence of recombinant YACs. This is illustrated in Fig. 2 after hybridization with an 8-kb EcoRI fragment (see Fig. 1B) containing the C22 and C)~3 genes (Hieter et al., 1981). In seven of the 20 colonies, a YAC of the predicted 120 kb size was identified (see Fig. 2A), and multiple digests confirmed that an authentic region including three V genes, seven J-C genes and the 3' enhancer was obtained in four clones. Detailed analysis of these clones, with one, cosC22 YAC, shown in Fig. 2B, identified an 80-kb NotI fragment and an 85-kb BssHII fragment which, upon double digestion, was reduced to 50 kb corresponding to the authentic region as illustrated in Fig. lB. Further digests with HindIII and EcoRI (Fig. 2C) were in agreement with

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Fig. 1. The human lg 2 light chain (IgL) locus on YACs. (A) A 380-kb region contains 28 V2 genes, seven C2 genes preceded by one J segment each and the 3' lg 2 enhancer (Frippiat et al., 1995). The V2 YAC accommodates 26 V2 genes whereas the J-C region with two additional V2 genes as well as the 3' enhancer (E) is cloned on three cosmids. (B) The three overlapping cosmids, designated N97B2, N59D10 and N78H4, are spanning a C2 region of l l 0 k b and were obtained from a chromosome 22-specific library constructed in Lawrist 16 (Frippiat et al., 1995). Overlapping regions of about 15 and 6 kb, which promote homologous assembly, are indicated by crosses. Ligation of the centromeric YAC arm, containing yeast specific sequences TRP1, ARS, CEN and the telomere (TEL), with cos N97B2 is shown on the left and ligation of cos N78H4 with the non-centromeric YAC arm, containing the URA3 gene and the telomere, is shown on the right. Yeast sequences are stippled. The C2 probe used for mapping, an 8-kb EcoRI fragment (Hieter et al., 1981), is indicated. Restriction sites for ligation, NotI, EagI, BssHII and MluI, are indicated and the diagnostic sites BssHII and NotI in the resulting recombinant cosC2 YAC are shown. Methods: Cosmid DNA was prepared as described (Stemmer, 1991) and for linearization, cos N97B2 and cos N59D10 were digested with EagI and cos N78H4 with BssHII. Ligation with respective dephosphorylated pYAC-RC (Marchuk and Collins, 1988) vector arms was performed at a 10 molar excess of vector DNA. The three cosmids were co-transfected into spheroplasted AB1380 yeast cells, grown in YPD (1% Bacto yeast extract, 2% peptone, 2% glucose, 50/~g/ml adenine sulfate), according to Burgers and Percival (1987), using 0.1 #g DNA of cos N97B2 ligated to the left YAC arm and 0.1 #g DNA of cos N78H4 ligated to the right YAC arm mixed with an excess of 0.3 #g DNA for the middle cosmid N59D10. DNA content was about 0.5 pg per 200 ml spheroplasts (approx. 6 x 10? cells) and, after transformation, cells were embedded in selective agar containing 1 M sorbitol and lacking uracil and tryptophan. The basic SD medium was as follows: YNB containing 2% glucose and 5 g/1 ammonium sulfate. For AB1380, this medium was supplemented with 50 mg/1 adenine sulfate, 20 mg/l histidine, 20 mg/1 tryptophan, 20 mg/1 lysine, 20 mg/1 uracil, 20 mg/l isoleucine and 30 mg/l threonine to give SD7. For YPH925 the minimal medium was supplemented with adenine sulfate, histidine, tryptophan, lysine, uracil at the same concentrations as above plus 60 mg/1 leucine to give SD6. Where appropriate, one or more aa or purine/pyrimidine bases were omitted from SD6 or SD7 to maintain selection of YACs (referred to as S D - ) . For solid medium agar was added to 2%. The yeast strains used have the following genotypes: AB1380 (MATa, [psi+], ura3-52, lys2. -1°care, ade2-1°cnre,his5"~'ber, trpl, canl-lO0 °~are, ile-, thr- ) and YPH925 (MATer, ura3-52,

lys2-801, ade2-101, his3A200, trplA63, leu2A1, cyh2 r, karlA15). p r e v i o u s r e s t r i c t i o n a n a l y s e s ( H i e t e r et al., 1981; T a u b et al., 1983; U d e y a n d B l o m b e r g , 1987; D a r i a v a c h et al., 1987; V a s i c e k a n d L e d e r , 1990; C o m b r i a t o a n d K l o b e c k , 1991) a n d h i g h l i g h t e d t h e c o r r e c t a s s e m b l y f r o m t h e recombined c o s m i d r e g i o n s w h i c h r e s u l t e d i n five internal fragments: a 4.6-kb double band, a 3.6-kb band and a 2.5-kb band from cosmid N59D10, and a ll-kb band from cosmid N78H4. The cosC22 YAC clone was further used for extension of the V2 YAC.

2.3. Transfer o f the V2 Y A C into a M A T s strain In order to allow homologous recombination lapping YACs they must be accommodated

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nucleus. Inefficient diploid formation can be achieved w h e n o n e o f t h e m a t i n g y e a s t cells c a r r i e s a k a r l z l l 5 m u t a t i o n ( H u g e r a t et al., 1994). T h e a d v a n t a g e is t h a t two haploid nuclei are combined in a heterokaryon allowing selection of haploid YAC ductants using the selectable marker genes provided by the parental strains. Since both overlapping YACs, V2 and cosC2, were cloned in the AB1380 M A T a host strain, this necessitated the transfer of one YAC into a M A T e strain in order to e x p l o i t t h e y e a s t s e x u a l cycle t o a l l o w efficient r e c o m b i nation of the two YACs. For the transfer of the V2 YAC i n t o t h e Y P H 9 2 5 M A T e s t r a i n , A B 1 3 8 0 cells c o n t a i n i n g the V2 YAC were grown under selective conditions and a b o u t 106 cells w e r e i n c u b a t e d w i t h e q u a l n u m b e r s o f

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Fig. 2. Analysis of recombination YAC assembly from cosmids. (A) DNA from cosmid transformants was analysed by Southern blots from PFGE hybridized with the C2-specific probe (see Fig. 1B). Lanes 1 11 show randomly picked yeast clones with two transformants, termed cosC22 YAC and cosC29 YAC in lanes 2 and 9, of the correct size of 120 kb. (B) Further analysis by PFGE and hybridization with the C2 probe, of undigested DNA and NotI and BssHII digests, shows the expected arm fragments of about 80 and 85 kb whilst the Nod and BssHII double digest of cosC22 YAC DNA shows the authentic internal fragment of about 50 kb. (C) Digests with EcoRI and HindIII highlight the internal C2 fragments obtained from the cosmids which are in agreement with previous results (Hieter et al., 1981; Taub et al., 1983; Udey and Blomberg, 1987; Dariavach et al., 1987; Ghanem et al., 1988; Vasicek and Leder, 1990). In the two authentic C2 YACs illustrated, cosC22 YAC and cosC29 YAC, identical bands of 2.5 kb, 3.6 kb and a 4.6-kb doublet were found; these contain C21, C22 and C23 and 4, respectively, and correspond to cos N59D10. An ll-kb band containing C25, 6 and 7 corresponds to cos N78H4 (Frippiat and Lefranc, unpublished observation). In other cosC2 YACs the bands were different or missing, as an example cosC211 is shown. Methods: (A) PFGE conditions were 0.5 x TBE, 1% agarose, 180 V, 10 s switch time for 27 h at 3.5°C. The PFGE size marker was 2 ladder. (B) PFGE conditions were 0.5 x TBE, 1% agarose, 170 V, 5 s switch time for 24 h followed by 3 s switch time for 8 h at 3.5°C. (C) DNA from cosC211 YAC, cosC22 YAC, cosC29 YAC, cosmid N59D10 and cosmid N78H4 was digested with HindIII and EcoRI and resolved on conventional agarose gels. 2 DNA digested with HindIII was used as the size marker.

Y P H 9 2 5 cells in Y P D for 6 h at 30°C. M a t e d cells were w a s h e d with w a t e r a n d p l a t e d on uracil- a n d t r y p t o p h a n deficient a g a r plates ( S D 6 - U r a - T r p ; see legend to Fig. 1) in the presence of 2 ~ g / m l c y c l o h e x i m i d e ( C Y H ) . This e n s u r e d t h a t only m a t e d cells survived as Y P H 9 2 5 does n o t g r o w w i t h o u t uracil a n d t r y p t o p h a n a n d AB1380 does n o t survive in the presence of C Y H . F u r t h e r tests to d i s c a r d rare d i p l o i d s ( H u g e r a t et al., 1994) s h o w e d the i n a b i l i t y of the clones to g r o w w i t h o u t histidine a n d leucine. T h e presence of the intact V2 Y A C in the h a p l o i d Y P H 9 2 5 strain was further verified by P F G E a n d h y b r i d i z a t i o n with specific V2 p r o b e s ( d a t a n o t shown). 2.4. YAC arm modifications to allow mitotic selection

Site-specific m o d i f i c a t i o n of YACs focused on two goals: to establish an efficient p o s i t i v e - n e g a t i v e selection p r o c e d u r e of m i t o t i c r e c o m b i n a t i o n events between two o v e r l a p p i n g YACs a n d the i n t e g r a t i o n of a selectable m a r k e r gene for s u b s e q u e n t i n t r o d u c t i o n of the YAC into m a m m a l i a n cells. In o r d e r to select for correct r e c o m b i n a t i o n events a n d a g a i n s t possible r e c i p r o c a l o r u n w a n t e d r e c o m b i n a t i o n p r o d u c t s , the cosC22 Y A C was site-specifically m o d i f i e d with p R A N 4 , a r e p l a c e m e n t t y p e v e c t o r ( M a r k i e et al., 1993). Site-specific i n t e g r a t i o n of this vector, shown in Figs. 3 a n d 4, resulted in the a d d i t i o n of the N E O gene a n d the d i s r u p t i o n of the

U R A 3 a u x o t r o p h i c m a r k e r in the acentric YAC a r m by i n t e g r a t i o n of ADE2, which allowed selection in adeninedeficient m e d i u m . As s h o w n in Fig. 3, these m o d i f i c a t i o n s resulted in YACs of the expected increased size (130 kb) when c o m p a r e d with the original YACs (120 kb). In a d d i t i o n , the p R A N 4 vector c o u l d also be found i n t e g r a t e d into a host yeast c h r o m o s o m e (Fig. 3, lane 6), a n d clones with two Y A C copies were identified (Fig. 3, lanes 4 a n d 5), a n o b s e r v a t i o n f o u n d b y us a n d others (Davies et al., 1992; M a r k i e et al., 1993); only one of t h e m was sitespecifically modified. To p r o v i d e i n d e p e n d e n t selection, the centric a r m of the V2 YAC was m o d i f i e d with p L N A , an i n t e g r a t i o n vector which site-specifically duplicates the amp gene, b y a d d i n g LYS2, to allow selection in lysine-deficient m e d i u m , a n d N E O ( D a v i e s et al., 1992). V2 Y A C yeast clones were o b t a i n e d which showed YACs m o d i f i e d with in t a n d e m copies of p L N A a n d e x t e n d e d to 300 k b in a d d i t i o n to the original YAC. F o r further YAC extension such a clone c o n t a i n i n g one V2 YAC with two copies of p L N A was t a k e n for h o m o l o g o u s r e c o m b i n a t i o n with a clone c o n t a i n i n g a single cosC22 Y A C e x t e n d e d to 130 k b by p R A N 4 . 2.5. Selection o f recombinant YACs

To r e c o m b i n e the two o v e r l a p p i n g YACs, AB1380 cells c o n t a i n i n g the cosC22 YAC m o d i f i e d with p R A N 4

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50-Fig. 3. cosC22 YAC modification with pRAN4. Southern blot hybridization with the 1.1-kb XhoI-SalI fragment of the NEO gene from pMC1NeoPolyA (Thomas and Capecchi, 1987). Lanes 1, 2 and 3 show yeast clones containing one copy of the cosC22 YAC modified with one copy of pRAN4 integrated into the right YAC arm. The integration of pRAN4 increased the size from about 120 kb to 130 kb. The cosC22 YAC yeast clones in lanes 4 and 5 contain two YAC copies, one modified with pRAN4. Such clones were able to grow in uracil- and adenine-deficient medium. In lane 6 pRAN4 is integrated into a host chromosome. The original cosC22 YAC is also highlighted by hybridization with the NEO probe due to corresponding sequences in the cosmid vector which are retained after linearization of cos N78H4. Methods: AB1380 yeast cells containing the cosC22 YAC were spheroplasted (Burgers and Percival, 1987) and 200 pl of cells were transformed with 1/~g pRAN4 digested with BamHI. Transformants were selected in SD7 - T r p - A d e containing 1 M sorbitol. DNA was prepared in blocks according to Rose et al. (1990) and PFGE conditions were 0.5 × TBE, 1.2% agarose, 180 V and 10 s switch time for 30 h at 3.5°C. w e r e m a t e d w i t h Y P H 9 2 5 cells c o n t a i n i n g the V2 Y A C m o d i f i e d w i t h p L N A as d e s c r i b e d in S e c t i o n 2.3). T h e r e c o m b i n a t i o n p r o d u c t s o b t a i n e d are s h o w n in Fig. 4 a n d Fig. 5. T o s u r v i v e s e l e c t i o n in S D - A d e -Lys m e d i u m , cells m u s t r e t a i n b o t h t h e p L N A m o d i f i e d V2 YAC and the pRAN4 modified cosC2 YAC. Although the t w o o v e r l a p p i n g Y A C s w h i c h a r e b r o u g h t t o g e t h e r in t h e n u c l e u s o f a h a p l o i d y e a s t cell c a n r e c o m b i n e w i t h e a c h o t h e r , t h e f r e q u e n c y of m i t o t i c r e c o m b i n a t i o n e v e n t s is n o t v e r y high. H e r e we u s e d a d o m i n a n t c o u n t e r - s e l e c t i o n o n 5 - F O A c o n t a i n i n g a g a r p l a t e s to d e t e c t cells w h i c h h a d r e c o m b i n e d b o t h Y A C s a n d discarded the smaller recombination product containing the f u n c t i o n a l U R A 3 g e n e ( B o e k e et al., 1984). I n t h a t way, f r o m a t o t a l of 100 clones, 14 w e r e a n a l y s e d by P F G E , o f w h i c h t h r e e s h o w e d t h e e x p e c t e d i n c r e a s e in size w h i c h r e s u l t e d in a Y A C o f 410 k b (see Figs. 4 a n d 5). As s c h e m a t i c a l l y i l l u s t r a t e d in Fig. 4, d e s i r e d (Fig. 4A) a n d u n d e s i r e d (Fig. 4B) r e c o m b i n a t i o n e v e n t s w e r e o b t a i n e d . S o u t h e r n b l o t a n a l y s i s o f P F G E in Fig. 5 ( l a n e s 4 a n d 5) s h o w s t h a t t h e 4 1 0 - k b r e c o m b i n a n t Y A C , t e r m e d V 2 - J C 2 Y A C , h y b r i d i z e d to b o t h the C 2 a n d N E O p r o b e s . F u r t h e r digests w i t h B s s H I I , N o t I a n d , s e p a r a t e l y , E c o R I a n d HindIII, f o l l o w e d b y h y b r i d i z a tion with the C2 probe (data not shown), confirmed that

B

20 kb YAC

Illegal Recombination ~ ¢ : = : ~ , v , ~ ~ - . < ~ L ~ ~ , .

2 8 0 k b V~. YAC

- ",,¢J'..,,~,.,'/.T¢=TI1#-.,7~/I - 130 kb cosC ~. Y A C

NEO ADE2

---~B~C:=',".clf~'.

90 kb Y A C

+ URA3

•~ ' , , ¢ / ~ - ! . ¢ ~

.

.

.

.

.

.

~

320 kb YAC

Fig. 4. Schematic representation of authentic and illegal recombination events between V2 YAC and cosC2 YAC. (A) The V2 YAC was modified with pLNA in the left YAC arm, which increased its size from 280 kb to 300 kb, to allow selection in lysine-deficient medium• The cosC2 YAC (clone 2) was extended by pRAN4 integration into the right YAC arm, which increased the size from 120 kb to 130 kb, to allow selection in adenine-deficient medium. Recombination between the approx. 15-kb 3' V2 YAC and 5' cosC2 YAC overlap (indicated by black boxes and cross) resulted in an authentic 380-kb region covering the V2-JC2 region on one 410-kb YAC termed V2-JC2 YAC. The V2-JC,~ YAC was selected in lysine- and adenine-deficient medium in the presence of 5-FOA. Reciprocal recombination resulted in a 20-kb YAC with the functional URA3 gene which is not maintained in 5-FOA. (B) Different recombination events can be identified in the same cell when the haploid state of the YACcontaining yeast cell is not maintained or when the yeast cell contains several differently modified YACs (see Fig. 3). Illegal recombination (indicated by lightly shaded boxes and cross) was found between the 280-kb unmodified V2 YAC and the pRAN4 modified 130-kb cosC2 YAC. It is likely that such undesired recombination was mediated by a putative hot spot of homology providing a recombination overlap 5' of V2 and 3' of C2. This resulted in a 90-kb YAC and a 320-kb YAC containing the URA3 gene which is removed by counterselection in 5-FOA.

the V 2 - J C 2 Y A C was in the e x p e c t e d c o n f i g u r a t i o n , as the o b t a i n e d r e s t r i c t i o n b a n d s w e r e i d e n t i c a l to the c o s C 2 2 Y A C a n d c o s m i d digests s h o w n in Fig. 2B a n d C. T h e r e c i p r o c a l r e c o m b i n a t i o n p r o d u c t , a n a p p r o x . 2 0 - k b Y A C , was d i s c a r d e d b y 5 - F O A s e l e c t i o n w h i c h is t o x i c in t h e p r e s e n c e o f a f u n c t i o n a l U R A 3 gene, in this i n s t a n c e p r o v i d e d by t h e u n m o d i f i e d r i g h t Y A C a r m . H o w e v e r , o t h e r r e c o m b i n a t i o n p r o d u c t s c a n be m a i n t a i n e d w h e n n o c o u n t e r - s e l e c t i o n is p r o v i d e d . W e r e p e a t e d l y o b s e r v e d a n u n e x p e c t e d o r 'illegal' r e c o m b i n a t i o n

200

A. ld Popovet al./Gene 177 (1996) 195.-201 1 2 3 4

1

2

3

4

5 kb m400

300

m 200

•

e

~0 ~

i~ mlO0

C~

NEO

Fig. 5. PFGE analysis of the V2 YAC and cosC2 YAC and their respective recombination products hybridized with C2 and NEO probes. In lane 1, the 130-kb cosC;t2 YAC modified with one copy of pRAN4 is highlighted in both hybridizations, whilst, shown in lane 2, the 300-kb V2 YAC modified with two copies of pLNA hybridized only with the NEO probe. Lane 3 illustrates the precursor recombination clone, containing both original YACs, the V2 YAC and the cosC22 YAC, before counter-selection with 5-FOA. In addition this clone also contained one copy of the unmodified V2 YAC (data not shown) which can participate in both correct and illegal recombination events (see Fig. 4). Lane 4 illustrates the authentic 410 kb V2-JC2 recombinant YAC hybridizing with both probes. Selection in lysineand adenine-deficient medium in the presence of 5-FOA achieved successful recombination and removed reciprocal recombination products with the exception of the 90-kb illegal recombinant YAC. Note that this YAC gives a weaker hybridization signal with the NEO probe as it only contains one copy of pRAN4 whilst the 410-kb V2-JC2 YAC contains two tandem copies of pLNA. Lane 5 shows a 410-kb V2-JC2 YAC clone after selective loss of the 90-kb illegal recombinant YAC. Methods: Recombination mating was performed as described in Section 2.3). After mating, cells were washed twice with water and grown on SD6 - A d e - L y s agar plates. The haploid status of the cells was verified by streaking the colonies on SD6 - H i s - L e u plates. To select for recombination events between two overlapping YACs the haploid yeast cells were plated on agar containing 1.7 g/l YNB without ammonium sulfate, 2% glucose, 1 g/1 proline as the sole nitrogen source, leucine, histidine and uracil (see Fig. 1 legend) and 100/~g/ml 5-FOA as supplement (McCusker and Davis, 1991). To remove the illegal 90-kb recombinant YAC, the URA3 gene was restored by transformation with the acentric YAC arm from pYAC-RC. Selection in uracil-deficient medium and subsequent counter-selection of streaked out colonies in 5-FOA and hybridization with the C2 probe identified clones which contained only the 410-kb V2-JC2 YAC. PFGE conditions were 0.5 x TBE, 1% agarose, 180 V, 40 s switch time for 15 h followed by 20 s switch time for 9 h at 10°C.

event due to a p u t a t i v e r e c o m b i n a t i o n hot spot in a region 5' of V2 a n d 3' of C2 (see Fig. 4B). As YACs have been f o u n d to be present in m u l t i p l e copies ( D a v i e s et al., 1992; M a r k i e et al., 1993), which w o u l d allow different r e c o m b i n a t i o n events, it was n o t surprising that two i n d e p e n d e n t r e c o m b i n a t i o n p r o d u c t s c o u l d be f o u n d in one cell. In t h a t w a y an a d d i t i o n a l 90-kb Y A C which

c o u l d not be r e m o v e d by 5 - F O A selection was identified after N E O h y b r i d i z a t i o n (see Fig. 5, lane 4). I n the e x p e r i m e n t s we o p t e d for m i t o t i c r e c o m b i n a t i o n in h a p l o i d cells because a t t e m p t s to use diploids s h o w e d n o t only a high frequency of illegal r e c o m b i n a t i o n events, b u t also resulted in YAC instability ( D e n D u n n e n et al., 1992). This instability was not seen in h a p l o i d cells, a l t h o u g h illegal a n d a u t h e n t i c r e c o m b i n a t i o n events o c c u r r e d at a similar frequency (see Fig. 4). T h e presence of yeast clones with m u l t i p l e YAC copies resulted in several r e c o m b i n a t i o n p r o d u c t s , of which one was usually correct. By a n d large the a p p l i e d selection p r o c e d u r e identified correct clones with the exception of the 90-kb n o n - a u t h e n t i c r e c o m b i n a t i o n YAC shown in Fig. 5 (lane 4). T h e presence of this 90-kb YAC was unlikely to cause any p r o b l e m s with the i n t r o d u c t i o n of the V2-JC2 YAC into e m b r y o n i c stem cells because after p r o t o p l a s t fusion usually only one c o p y of a YAC integrates into the g e n o m e of m a m m a l i a n cells (Davies et al., 1992). H o w e v e r , we o p t e d for the r e m o v a l of the illegal YAC as described by H e i k o o p et al. (1994). This was easily achieved by t r a n s f o r m a t i o n with the acentric YAC a r m of p Y A C - R C to replace the p R A N 4 m o d i f i e d right arm. I n t e g r a t i o n by h o m o l o g o u s r e c o m b i n a t i o n into either the V2-JC,~ YAC or the 90-kb chimeric YAC restores the U R A 3 gene a n d allows selection in uracil-deficient m e d i u m . As s h o w n in Fig. 5 (lane 5), counter-selection in 5 - F O A r e m o v e d the 90-kb r e c o m b i n a t i o n YAC and, thus, yeast clones c o n t a i n i n g a single m o d i f i e d V2-JC2 YAC were o b t a i n e d .

3. Conclusions In o r d e r to assemble a c o m p l e x locus in a u t h e n t i c c o n f i g u r a t i o n we have d e v e l o p e d a new strategy to c o n s t r u c t YACs from m u l t i p l e o v e r l a p p i n g c o s m i d clones. This a p p r o a c h was c o m b i n e d with a simple a n d efficient genetic screening m e t h o d for m i t o t i c r e c o m b i n a tion events between two YACs with different selectable m a r k e r genes i n t r o d u c e d into their arms. A high frequency of successful r e c o m b i n a t i o n of 6- a n d 15-kb overlaps resulted in a 410-kb YAC c o n t a i n i n g a contiguous 380-kb germline region of the h u m a n 2 light chain locus h a r b o u r i n g 28 V2 genes, seven J2-C2 regions a n d the 3' enhancer. T h e r e - a s s e m b l y of this c o m p l e x locus a n d the site-specific i n t e g r a t i o n of a m a m m a l i a n selectable m a r k e r gene m a k e s it possible to transfer this YAC into e m b r y o n i c stem cells which m a y give rise to transgenic mice ( Z o u et al., 1996) essential for structurefunction studies of the h u m a n Ig2 locus.

Acknowledgement We t h a n k Drs. D. M a r k i e for the k i n d gift of p R A N 4 a n d L. B o w d e n for helpful c o m m e n t s on the m a n u s c r i p t .

A. ld Popovet al./Gene 177 (1996) 195-201 We are particularly indebted to Dr. Greg Winter for the generous provision of mapping results prior to publication and his advice in planning the experiments. This work was supported in part by the Biotechnology and B i o l o g i c a l S c i e n c e s R e s e a r c h C o u n c i l (A.V.P., M.B.), t h e M R C (C.B., M . B . ) a n d c o r e f u n d i n g f r o m t h e B a b r a h a m Institute.

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