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Investigation of Schizosaccharomyces pombe as a cloning host for human telomere and alphoid DNA
Gene 241 (2000) 275–285 www.elsevier.com/locate/gene
Investigation of Schizosaccharomyces pombe as a cloning host for human telomere and alphoid DNA Kathryn L. Mann, Clare Huxley * Section of Molecular Genetics, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, London SW7 2AZ, UK Received 23 August 1999; received in revised form 17 October 1999; accepted 29 October 1999 Received by V. Larionov
Abstract The fission yeast Schizosaccharomyces pombe (Sch. pombe) has been proposed as a possible cloning host for both mammalian artificial chromosomes (MACs) and mammalian genomic libraries, due to the large size of its chromosomes and its similarity to higher eukaryotic cells. Here, it was investigated for its ability to form telomeres from human telomere sequence and to stably maintain long stretches of alphoid DNA. Using linear constructs terminating in the telomere repeat, T AG , human telomere 2 3 DNA was shown to efficiently seed telomere formation in Sch. pombe. Much of the human telomeric sequence was removed on addition of Sch. pombe telomeric sequence, a process similar to that described in S. cerevisiae. To investigate the stability of alphoid DNA in fission yeast, bacterial artificial chromosomes (BACs) containing 130 and 173 kb of alphoid DNA were retrofitted with the Sch. pombe ars1 element and ura4+ marker using Cre-lox recombination. These alphoid BACs were found to be highly unstable in Sch. pombe deleting down to less than 40 kb, whilst control BACs of 96 and 202 kb, containing non-repetitive DNA, were unrearranged. Alphoid DNA has been shown to be sufficient for human centromere function, and this marked instability excludes Sch. pombe as a useful cloning host for mammalian artificial chromosomes. In addition, regions containing repetitive DNA from mammalian genomes may not be truly represented in libraries constructed in Sch. pombe. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Bacterial artificial chromosome; Cre recombinase; Mammalian artificial chromosome; Schizosaccharomyces pombe artificial chromosome library
1. Introduction The field of mammalian artificial chromosome (MAC ) research has recently been stimulated by the generation of minichromosomes by truncation of preexisting chromosomes or by transfection of alphoid DNA into human cells (Harrington et al., 1997; Shen et al., 1997; Ikeno et al., 1998; Mills et al., 1999). The generation of a MAC provides not only an immediate opportunity to study the elements required for chromosome structure and function but also the possibility of Abbreviations: ars, autonomously replicating sequence; BAC, bacterial artificial chromosome; Cm, chloramphenicol; Km, kanamycin; MAC, mammalian artificial chromosome; NR, DNA non-resolving DNA; PFG, pulsed-field gel; SPARC, Schizosaccharomyces pombe artificial chromosome; TAS, telomere associated repeats. * Corresponding author. Tel.: +44-207-594-3028; fax: +44-207-594-3015. E-mail address: [email protected] (C. Huxley)
developing new vectors for gene therapy and for making transgenic animals. A stable, segregating, linear chromosome requires three functional components; centromeres, telomeres and origins of replication. Alphoid DNA has been shown to be sufficient for centromere formation after transfection into HT1080 cells ( Harrington et al., 1997; Ikeno et al., 1998; Henning et al., 1999), telomere formation requires hundreds of bp of the telomere repeat T AG (Hanish et al., 1994), and 2 3 large fragments of DNA are able to replicate extrachromosomally in mammalian cells (Heinzel et al., 1991; McGuigan and Huxley, 1996). A useful MAC cloning host should allow the propagation of the intact MAC, modification of the MAC (probably by efficient homologous recombination) and transfer of the MAC into other cells where it would be maintained in an unrearranged form. E. coli has been used to clone the alphoid DNA arrays used for minichromosome formation by transfec-
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tion into HT1080 cells ( Harrington et al., 1997). The resulting minichromosomes were far larger than the input DNA, which must have recombined during chromosome formation. This experiment demonstrated that alphoid DNA is stable in E. coli with arrays of 173 kb of alpha satellite in BAC vectors being maintained upon extended growth. However, E. coli currently has a cloning capacity of up to 300 kb, which is limited by the inability to introduce larger DNA molecules into cells intact. In contrast, the smallest mammalian chromosomes are still 2.4 Mb in size (Mills et al., 1999). S. cerevisiae has also been used to clone alphoid DNA prior to transfection into HT1080 cells (Ikeno et al., 1998; Henning et al., 1999). Again, the input DNA was rearranged during minichromosome formation. S. cerevisiae’s main drawback as a MAC cloning host is that arrays of centromeric satellite DNA much larger than 100 kb are unstable (Neil et al., 1990; McGuigan et al., 1998). This is due to the efficient recombination of the tandemly repetitive DNA into smaller arrays. Alternative yeast strains deficient in genes involved in recombination, including rad52, have proved to be more stable cloning hosts for repetitive DNA, although the arrays cloned in these hosts are still relatively small (Neil et al., 1990; Ikeno et al., 1998; McGuigan et al., 1998). A more promising cloning host is the chicken DT40 cell line. Chickens and mammals share telomeric sequences, mammalian chromosomes segregate accurately in DT40 cells (Shen et al., 1997), and, importantly, this cell line shows high rates of recombination between chromosomal DNA and homologous exogenous DNA (Buerstedde and Takeda, 1991). A limitation is that the techniques that have been developed to transfer mini chromosomes between DT40 and other cell lines, such as microcell fusion, are inefficient and technically difficult. Purification of the MAC construct from DT40 cells will always be inefficient as they are megabase molecules maintained at single copy, and the host genome is large. Finally, Sch. pombe, a fission yeast, is a potential MAC cloning host. Its endogenous chromosomes possess several promising features compared to those of S. cerevisiae. Its largest chromosome is 5.7 Mb (as opposed to about 1.6 Mb in S. cerevisiae), indicating that it could accommodate MAC-sized DNA. Its centromeres are also more similar to those of higher eukaryotes, consisting of 35–110 kb of untranscribed, repetitive DNA with the repetitive elements arranged in both inverted and tandem formations around a non-repetitive central core (Steiner et al., 1993). Furthermore, Sch. pombe’s telomere associated sequence ( TAS ) exhibits a satellitelike arrangement with 86–89 bp repeating units forming higher order repeats of 0.9–1.2 kb, which in turn form arrays totalling at least 19 kb in length (Sugawara, 1989). These features suggest that the stability of repeti-
tive DNA may be greater in fission yeast than in S. cerevisiae. Sch. pombe artificial chromosome (SPARC ) vectors have been developed, containing Sch. pombe telomeric sequences ( T AC A C G ) (Sugawara and 2 0–1 0–1 0–1 1–8 Szostak, 1986), an autonomously replicating sequence (ars1) for replication and selectable markers (Nimmo et al., 1994). These vectors have been used to successfully clone human DNA in Sch. pombe in a linear form ( Young et al., 1998); 50 kb cosmids were subcloned without rearrangements or circularization. Preliminary experiments to investigate the possibility of cloning NotI-digested genomic DNA generated clones containing SPARCs of between 80 and 450 kb with a copy number of between five and 10 copies per cell. These molecules were found to be relatively stable upon extended growth ( Young et al., 1998), indicating that this system could be used to generate genomic libraries of mammalian DNA. However, the stability of repetitive DNA in Sch. pombe was not investigated. The ability of human telomeric DNA to seed telomeres in Sch. pombe would allow a MAC to be maintained in a linear form when transferred from mammalian cells to Sch. pombe and would possibly allow it to be shuttled back into mammalian cells directly. Transfer of a modified 3.5 Mb Sch. pombe chromosome into a mouse cell line using cell-to-cell fusion has previously been demonstrated (Allshire et al., 1987). Both Tetrahymena (Sugawara and Szostak, 1986; Shervington et al., 1993) and S. cerevisiae (Guerrini et al., 1985; Hahnenberger et al., 1989) telomeric repeats have been shown to seed telomeres in Sch. pombe, though the efficiencies of telomere formation vary considerably, from 5 to 100%, between studies. Here, we investigate the capacity of human telomere sequence to seed telomere formation in Sch. pombe and the stability of alphoid DNA in Sch. pombe.
2. Materials and methods 2.1. Schizosaccharomyces pombe host strain The Sch. pombe strain FY562 (h+leu1-32ura4D18rec36-55) was used in all experiments. This strain exhibits a 10-fold reduction in intragenic mitotic recombination compared to the wild-type strain (GyslerJunker et al., 1991). In S. cerevisiae, tandemly repeated DNA such as Y chromosome alpha satellite has been shown to be more stable in the recombination-deficient rad52 strain (Neil et al., 1990; McGuigan et al., 1998). The rad52 strain is also deficient in intragenic recombination ( Kouprina et al., 1994), and thus it was thought that the FY562 strain might provide a more stable cloning environment for alphoid DNA than wild-type Sch. pombe.
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2.2. Plasmids and cloning The plasmids pTF2 (Tolmachova et al., 1999) containing 800 bp of human telomeric sequence, and pEN51 (Nimmo et al., 1994), containing 258 bp of Sch. pombe telomere repeats and 800 bp of TAS, were used in the telomere study. DNA was gel-purified using the Qiaex II gel purification kit (Qiagen). DNA ligase, calf intestinal alkaline phosphatase and all restriction enzymes were obtained from Gibco BRL. To construct the pTF2:pEN51 molecule, pEN51 was digested with SacI, dephosphorylated and digested with XhoI to give a 6.6 kb fragment. pTF2 was linearized with NotI, dephosphorylated and digested with SalI to give an 8.9 kb fragment with a complementary site to XhoI. Following gel purification, the fragments were ligated, and the correct 15.5 kb molecule (pTF2: pEN51) was isolated from non-ligated pTF2 and pEN51 arms, and from pTF2:pTF2 and pEN51:pEN51 ligation products by gel purification. The BAC retrofitting plasmid, pBLTK.ars1.ura4+, was made in the following way. pBT was made by cloning a 2.4 kb XmnI fragment from pBR322 (New England Biolabs) carrying the tetracycline resistance gene (Tcr) into the HincII site of pBluescript II SK (Stratagene). A 0.8 kb SmaI/HindIII fragment from pBS226 (Gibco BRL) containing the loxP site was cloned into the SmaI/HindIII sites of pBT to form pBLT. A 1.2 kb SacI fragment from pEN51 carrying the kanamycin resistance gene (kmr) was then inserted into the SacI sites of pBLT to give pBLTK. Finally, a 3.6 kb PvuII/XmnI fragment from pEN51 containing the Sch. pombe ars1 and ura4+ marker was cloned into the EcoRV site in the Tcr gene of pBLTK, to give pBLTK.ars1.ura4+. 2.3. Analysis of termini pTF2:pEN51 DNA was isolated from a Seaplaque low melting point agarose ( FMC ) pulsed field gel using beta-Agarase I (New England Biolabs) and recovered by ethanol precipitation. This DNA was used in the BAL-31 nuclease analysis. The DNA was digested with 0.01, 0.1, 0.3, 0.6, 1.0 and 3.0 U of BAL-31 for 20 min, at which point, the reaction was terminated by the addition of EGTA. The DNA was extracted with phenol/chloroform, precipitated with ethanol and digested with BglII. To clone the terminus prior to sequencing, the 3∞ G-rich telomere overhang was removed with varying amounts of BAL-31 nuclease (New England Biolabs) applied for 30 min at 30°C. The reaction was terminated with EGTA, and the DNA was reprecipitated and bluntended with T4 DNA polymerase (New England Biolabs). A terminal fragment was generated by KpnI digestion and cloned into SmaI/KpnI-cut pBluescript.
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Colony hybridization was used to identify clones carrying a human telomere insert. Colonies were inoculated in 96 well plates and transferred to Hybond-N filters placed on LB plates containing ampicillin using a microplate replicator. These were incubated at 37°C overnight. The filters were then prepared by standard methods and hybridized as described below with a human telomere probe. Positive colonies were sequenced using the M13F and M13R primers (Stratagene) and Amersham Life Sciences Sequenase Version 2.0 kit, following the manufacturer’s instructions. 2.4. Sch. pombe transfections, screening and DNA preparation The protocol described by Allshire (1992) was followed except for the replacement of the spheroplasting enzyme NovoZym 234 by Zymolyase 100T (ICN ). This enzyme is partially purified by affinity chromatography, contains no RNase or DNase activity and consistently results in higher transfection and faster colony growth. Between 1.5 and 2.5×108 cells were incubated with 6 mg of Zymolyase 100T at 37°C until 70–90% of cells had spheroplasted. For colony hybridization, Sch. pombe colonies were streaked on the appropriate plates and incubated at 30°C for 3 days. Hybond N filters (Amersham) were placed on the plates, which were incubated for a further 18 h. The filters were then placed in SP2 (1.2 M sorbitol, 50 mM sodium citrate, 50 mM sodium phosphate, pH 5.6) (Allshire, 1992)/b-mercaptoethanol (1 ml/ml SP2) for 15 min, followed by an overnight incubation at 37°C in SP2/b-mercaptoethanol/Zymolyase 100T (200 mg/ml ). Once 70–90% of cells had spheroplasted, the filters were treated, as described in Amersham’s instructions, and hybridized with the neor gene probe, as described below. Agarose plugs were prepared for the isolation of intact DNA. Cells were spheroplasted (Allshire, 1992), resuspended at a concentration of 2×109 cells/ml and mixed with an equal volume of 2% LMP agarose solution. This suspension was aliquoted into pre-chilled plug moulds and allowed to set. The plugs were incubated in a 1% LDS solution (1% dodecyl lithium sulphate, 100 mM EDTA, 10 mM Tris–Cl, pH 8.0) at 37°C for 1 h with gentle shaking, followed by a further incubation in 1% LDS, overnight at 37°C. The plugs were then washed twice for 2 h in 20% NDS (0.2% Nlauryl sarcosine, 100 mM EDTA, 2 mM Tris, pH 9.0) at room temperature and stored in this solution at 4°C. 2.5. BAC clones and DNA preparation The alphoid BACs, pBAC17a48 and pBAC17a64 were a gift of J. Harrington and contain 129.6 and 172.8 kb of pure alphoid DNA from chromosome 17,
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respectively (Harrington et al., 1997). The control BACs, L269 and L281, were a gift of B. Birren and are 96 and 202 kb, respectively. They were derived from chromosome 17, and sequence information is available from the Whitehead Institute/MIT Genome Sequencing Project. BAC DNA was prepared using a standard alkaline lysis miniprep method including a phenol/chloroform extraction.
telomere probe was a 0.8 kb NotI/SstI fragment from pSXneo-0.8-T AG (Hanish et al., 1994). Alphoid con2 3 sensus probe was amplified from total human genomic DNA using the a27 and a30 primers (Dunham et al., 1992). Finally, DNA from Caco 2 cells (ATCC ) was used as the total human DNA probe. These probes were labelled using the Megaprime DNA labelling system (Amersham), and unincorporated nucleotides were removed using NucTrap purification columns (Stratagene).
2.6. Retrofitting of BAC clones using the Cre-lox system Cre recombinase was a kind gift from P. Sadowski (Shaikh and Sadowski, 1997). Plasmid DNA was mixed with BAC DNA at equal molar concentrations and a total mass of between 100 and 500 ng. Ten times Cre reaction buffer (Novagen), 1 ml of Cre recombinase (3.6 mg/ml ) and H O were added to give a total reaction 2 volume of 30 ml which was incubated at 37°C for 1 h. The reaction was terminated by a phenol/chloroform extraction. DNA was recovered by ethanol precipitation and resuspended in 10 ml of H O. Two microlitres were 2 electroporated into 20 ml aliquots of ElectroMAX DH10B cells (Gibco BRL) at 1.4 kV, 100 V, 25 mF using a Bio-Rad Gene Pulser. 2.7. DNA analyses Agarose plugs of high-molecular-weight DNA were washed twice for 2 h in TE buffer at room temperature to remove the NDS. They were then equilibrated in the appropriate enzyme buffer for 2×30 min. The plugs were digested in 1× restriction buffer with 100 mg/ml of bovine serum albumin and between 40 and 60 U of restriction enzyme, overnight, at the recommended temperature. Between 0.5 and 1 mg of BAC DNA was digested for 2 h with 5–10 U of enzyme at the appropriate temperature. Pulsed field gel electrophoresis (PFGE ) was performed on a BIO-RAD CHEF-DRIII system. DNA was separated on 1% agarose gels, run in 0.5× TBE, and then transferred to Hybond N+ membrane (Amersham), as described by the manufacturer. Both the prehybridization and hybridization steps were carried out in Church buffer (16.8 g/l of NaH PO .H O, 2 4 2 54.1 g/l of Na HPO .12H O, 7% SDS, 100 mg/ml 2 4 2 salmon sperm DNA) at 65°C. Filters were washed in 2–0.5× SSC (20×: 175.3 g/l of NaCl, 88.2 g/l of sodium citrate, pH 7.0)/0.1% SDS at 65°C. The neor gene probe was a 1.1 kb XhoI/SalI fragment obtained from pMC1NeoPolyA (Strategene) The ura4+ gene probe (1.8 kb) and the ampr gene probe (2.1 kb) were isolated from pEN51 using a PvuII/ClaI digest. The LYS2 gene was a 5 kb EcoRI/HindIII fragment from pCH37, a plasmid derived from YCp50 with the LYS2 gene from YIp333 cloned into it. The human
3. Results 3.1. Human telomere function in Sch. pombe A linear molecule, pTF2:pEN51, was constructed from the pTF2 and pEN51 plasmids, to terminate at one end in 258 bp of Sch. pombe telomeric sequence and at the other with 800 bp of human telomeric sequences ( Fig. 1). This molecule was used to assess the telomere seeding efficiency of human telomeric sequence in Sch. pombe. To verify the human telomere sequence in pTF2, the 0.8 kb NotI/KpnI telomere fragment from the original telomere plasmid, pSXneo-0.8-T AG (Hanish et al., 2 3 1994), was subcloned and sequenced (Fig. 2). The sequence was unexpectedly found to be a complex mixture of human T AG and T G telomeric sequences, 2 3 2 4 with T AG arrays contributing 62% of the 654 bp 2 3 sequenced. A reasonable explanation for this is that the complex array may have been present in the human telomere from which this fragment was cloned. Mixed arrays of T AG , T G and TGAG sequences have 2 3 2 4 3 previously been found close to the terminal blocks of T AG in some human chromosomes (Allshire et al., 2 3 1989). This 800 bp sequence does seed efficient formation of telomeres in mammalian cells (Hanish et al., 1994; Tolmachova et al., 1999). The linear pTF2:pEN51 DNA molecules were transfected into Sch. pombe, and the pEN51 arm was selected for on ura− plates. Colony hybridization using a neor probe identified clones carrying the pTF2 arm.
Fig. 1. Map showing the linear pTF2:pEN51 construct. The molecule terminates in 258 bp of Sch. pombe telomeric sequence and 800 bp of human telomeric sequence and contains a ura4+ gene for selection purposes and an ars1 element for replication in Sch. pombe.
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Fig. 2. Partial sequence of the 0.8 kb human telomere fragment of pSXneo-0.8-T AG . The KpnI/NotI fragment was subcloned into pBluescript 2 3 and then sequenced using M13F and M13R primers. The M13F primer produced 222 bp of sequence from the KpnI end of the telomere. The M13R primer produced 414 bp of sequence from the NotI end of the human telomere fragment, representing the sequence at the very terminus of the pTF2:pEN51 molecule. Human T AG repeats are in bold. T G and other repeats are in normal type. 2 3 2 4
Ten clones were chosen for further analysis; six clones showing strong hybridization to the neor probe (clones 1–6), one control clone containing the original pEN51 plasmid (clone 7) and three clones showing no hybridization to the neor probe (clones 8–10). Pulsed field gel analysis and Southern blotting revealed that all the clones contained extrachromosomal elements (Fig. 3A). Clones 1–6, 8 and 10 exhibited bands corresponding to the linear 15.5 kb pTF2:pEN51 molecule when hybridized with neor, ura4+ and human telomere probes. Clones 8 and 10 were presumably false negatives from the colony hybridization due to inconsistent hybridization of the neor probe. Clone 7 contained the 9.1 kb circular pEN51 molecule and hybridized only to the ura4+ probe; the supercoiled molecules run more slowly than linears on this PFG, and the intense hybridization at the well is due to trapped open circular molecules. Clone 9 contained a 12 kb molecule, which again hybridized only to the ura4+ probe and is probably a linear pEN51:pEN51 molecule. To confirm that the molecules were linear, clone 5 was analysed after treatment with BAL-31 nuclease, an exonuclease that degrades both 3∞ and 5∞ termini of DNA, and BglII ( Fig. 3B). Hybridization with a LYS2 probe identified the pTF2 terminus, which exhibits a progressive reduction in size with increasing amounts of BAL-31 nuclease, indicating that it is a linear molecule. Hybridization with an ampr probe identified both the pEN51 terminus, again decreasing in size, and a constant band representing an internal fragment undigested by BAL-31 nuclease (data not shown). The telomere seeded in Sch. pombe by the human telomeric sequence on pTF2 was sized using a KpnI digest and Southern analysis (data not shown) and found to be heterogeneous, both within and between clones. Each clone was found to have a similar range of telomere size heterogeneity of 150–200 bp. However, the
average telomere length varied between clones with clone 2 having 220–420 bp, clone 3 having 380–600 bp and clone 5 having 300–450 bp of telomere array. To identify the length of the human telomeric sequence remaining at the terminus and to investigate the addition of Sch. pombe’s own telomeric repeats, the human telomere end of clone 5 was subcloned into pBluescript and sequenced. Two clones containing different human:Sch. pombe sequence junctions were identified. In one, Sch. pombe telomeric repeats are directly attached to 186 bp of the human telomere sequence disrupting a T AG array to form a 2 3 T AG :T ACACG junction (Fig. 3C ). No mixing of 2 3 2 2 the Sch. pombe and human arrays was observed, and the junction itself does not disrupt either repeat. Therefore, 614 bp of the human telomere array were removed during the addition of Sch. pombe telomeric sequence. As the telomere array from the KpnI site is between 300 and 450 bp in this clone, between 100 and 250 bp of Sch. pombe sequence have been added to the remaining 186 bp of human telomere sequence to form a functional telomere. The other clone was found to have a T G :T ACACG junction (data not shown), 2 4 2 2 with the Sch. pombe telomere attached to 216 bp of the human telomeric sequence. As the pTF2:pEN51 molecule terminates at one end in Sch. pombe telomeric repeats, the possibility that these were involved in the telomere formation process at the human telomeric terminus, perhaps by homologous recombination, was investigated. In similar experiments in S. cerevisiae, it has been demonstrated that recombination between the terminal sequences does occur prior to capping with host telomeric sequence (Pluta and Zakian, 1989). A linear molecule was constructed, terminating at both ends in 800 bp of human telomere and carrying the Sch. pombe ura4+ marker and ars1. Following transfection into Sch. pombe and pulsed
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Fig. 3. Analysis of Sch. pombe clones carrying pTF2:pEN51 molecules. (A) High-molecular-weight DNA from each of the 10 clones and the original FY562 strain of Sch. pombe were separated on a pulsed field gel, with 2–5 s switching, for 15 h at 14°C. The gel was Southern-blotted and the filter hybridized with the ura4+ probe. All clones contain extrachromosomal elements: clones 1–6, 8 and 10 exhibit DNA bands running at about 15.5 kb (although the bands in clones 6 and 10 are only visible with a longer exposure); clone 7 contains a circular pEN51 molecule, whilst clone 9 shows a band running at about 12 kb. The positions of the wells, the non-resolving DNA (NR) and the size markers are indicated at the left of the gel. (B) Extrachromosomal pTF2:pEN51 molecule from clone 5 was treated with varying amounts of BAL-31 nuclease as shown above each lane. The DNA was subsequently digested with BglII and separated on an agarose gel, which was Southern-blotted and hybridized with a LYS2 probe to identify the 4.7 kb human telomere arm. The gradual reduction in size of this fragment with increasing amounts of BAL-31 nuclease identifies this as a terminus, and thus the pTF2:pEN51 molecule as linear. The positions of size markers and the sizes of fragments are indicated on the left. (C ) Termini of the pTF2:pEN51 molecules from clone 5 were cloned and sequenced. The sequence shown illustrates the junction between human (T AG ) and Sch. pombe ( T AC A C G ) telomeric repeats. The mixed T AG and T G repeats are part of the complex 2 3 2 0–1 0–1 0–1 1–8 2 3 2 4 pattern of the human telomere fragment.
field gel analysis, three of seven clones carrying both arms were found to be linear and of the correct size (data not shown). As this two-armed molecule was not purified prior to transfection, a range of ligated and non-ligated molecules would also have been introduced into Sch. pombe, probably accounting for the lower frequency of correctly sized molecules observed here. However, this experiment does confirm the efficient
seeding of telomeres in Sch. pombe by human telomeric sequence, probably independent of intramolecular recombination. 3.2. Retrofitting BACs in vitro using Cre-lox In order to investigate the stability of large arrays of alphoid DNA in Sch. pombe we used two BAC clones,
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pBAC17a48 and pBAC17a64, which contain 130 and 173 kb of alphoid, respectively (Harrington et al., 1997). Alphoid BACs, rather than YACs, were chosen to investigate the stability of large alphoid arrays in Sch. pombe as circular molecules do not require telomere formation for extrachromosomal maintenance, and alphoid arrays of over 100 kb in size are reasonably stable in bacteria. For extrachromosomal maintenance in Sch. pombe, the BACs first had to be retrofitted with a marker, in this case, ura4+, and with an ars1 element. Initial attempts to sub-clone the BAC insert into a new vector containing these necessary elements were unsuccessful. Therefore, an alternative strategy was designed, exploiting the loxP site present in all BAC vectors. Here, Cre recombinase promotes site specific recombination between the loxP site on the BAC vector and a loxP site on the retrofitting plasmid, resulting in the insertion of relevant DNA on to the BAC, as shown in Fig. 4. A similar procedure has recently been published ( Kim et al., 1998). Four BACs were retrofitted, pBAC17a48 and pBAC17a64 containing alphoid DNA, and L269 and L281 containing 96 and 202 kb of single-copy sequence from chromosome 17, respectively. After mixing the BAC DNA with the retrofitting construct and Cre protein, the DNA was electroporated into E. coli. Colonies were selected for dual resistance to chloramphenicol (Cm) and kanamycin ( Km), to select for the BAC and retrofitting molecule, respectively. As the BssHII religated fragment lacks an origin of replication, it is unable to exist episomally and can only be maintained by integrating into the BAC or bacterial genome. BACs were analysed by restriction digestion and pulsed field gel analysis (data not shown). All were found to have the retrofitting DNA correctly inserted into the BAC loxP site. All of the retrofitted control BAC clones that were analysed were found to be of the correct size, whilst three out of 11 of the 130 kb alphoid BACs and two out of three of the 173 kb alphoid BACs were found to have deleted down to a smaller size. 3.3. Alphoid DNA stability in Sch. pombe The retrofitted alphoid and control BACs were transfected into Sch. pombe, and colonies were selected on ura− plates. Clones were analysed by restriction digestion, pulsed field gel and Southern analysis (Fig. 5). From the transfections of the alphoid BACs, 10 pBAC17a48 and four pBAC17a64 clones were analysed, and in all of them, the alphoid DNA was found to have deleted down to between 3 and 40 kb in size. Fig. 5A shows the 10 pBAC17a48 clones that have all deleted to less than 50 kb. Furthermore, three of the alphoid clones exhibit more than one band. Fig. 5B shows three of the pBAC17a64 clones, where the 173 kb input alphoid has been reduced to between 10 and 40 kb. The
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Fig. 4. Illustration of the BAC retrofitting strategy. Construction of the BAC retrofitting plasmid, pBLTK.ars1.ura4+, is described in Section 2.2. It carries the ura4+ and ars1 elements, a loxP site and a kmr marker for selection in bacteria. In order for the retrofitted BAC to be retained at single copy, the retrofitting plasmid’s origin (ColE1) must be removed prior to the Cre-lox reaction. This was achieved with a BssHII digestion of pBLTK.ars1.ura4+, which separates the ori and ampr backbone from the multiple cloning site containing the relevant elements for insertion onto the BAC. The appropriate BssHII fragment was gel-purified, religated and used in the Cre-lox reaction with BAC miniprep DNA. Cre recombinase promotes site specific recombination between the two loxP sites, resulting in the insertion of the ars1, ura4+ and kmr elements onto the BAC. As the religated BssHII fragment lacks an origin of replication, it is unable to exist episomally and can only be maintained by integrating into the BAC or bacterial genome. Therefore, following transfection, clones carrying the retrofitted BAC were selected for using double Km/Cm selection. All analysed clones contained a retrofitted BAC.
control BACs, L269 and L281 were also transfected into Sch. pombe, and five L269 clones and four L281 clones were analysed ( Fig. 5B). All were stable with the exception of one of the 202 kb L281 BACs, which had deleted to approximately 100 kb. A slight growth advantage displayed by clones containing alphoid DNA compared to those containing non-alphoid DNA was noted. Furthermore, two Sch. pombe proteins with homology to the mammalian alphoid-binding protein, CENP-B, have been identified,
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Fig. 5. Analysis of alphoid BACs in Sch. pombe. Agarose plugs of DNA were prepared from ura4+ Sch. pombe clones and digested with SalI or NotI to remove the BAC vector. The BAC inserts were resolved on a pulsed field gel, with 5–20 s switching for 20 h at 14°C. The gels were Southern blotted and the filters hybridized with an alphoid probe or total human DNA. (A) pBAC17a48 molecules in 10 Sch. pombe clones have deleted to less than 50 kb in size. The lane labelled ‘a YAC’ contains a 140 kb YAC containing alphoid DNA. (B) pBAC17a64 molecules in the 3 Sch. pombe clones have deleted to between 10 and 35 kb in size in comparison to the original 180 kb BAC run in the lane labelled ‘c’. Three of the L281 clones are unrearranged and run at the same size as the original 200 kb BAC run in the lane labelled ‘c’, while one clone has deleted to about 100 kb. All five of the L269 clones are unrearranged and give the same three SalI fragments as the original BAC in the lane labelled ‘c’. The positions of the wells and the NR DNA and the sizes of fragments are indicated on the left of the gel.
Abp1p and Cbh (Halverson et al., 1997; Lee et al., 1997). As the binding sites of these two proteins have not been fully characterized, we investigated the possibility that these proteins are binding to the alphoid DNA to give the molecules a segregational advantage in Sch. pombe. A mitotic stability assay (Hahnenberger et al., 1989) was carried out on clones containing approximately 40 kb of alphoid and those containing non-alphoid DNA. No difference was observed between the rate of loss of these molecules per generation, with both types being less stable than the pEN51 plasmid. An alternative explanation for the growth difference is that the non-alphoid molecules are generally larger due to their being unrearranged in Sch. pombe. Consequently, replication of these molecules may take longer and cause a slight delay in the cell cycle.
4. Discussion The data presented here show that human telomeric sequence can efficiently seed telomere formation in Sch. pombe. Following transfection into Sch. pombe of a 15.5 kb linear molecule terminating at one end with human telomere sequence, all eight of the neo and ura4+ positive clones that were analysed were found to
be linear. The two human:Sch. pombe telomere sequence junctions found may have been generated from separate telomere seeding events in two molecules or dynamic changes in telomere length in just one molecule that had already been stabilized by the addition of Sch. pombe telomeres. It was also demonstrated that a substantial proportion of the 800 bp of human telomere sequence is removed concomitantly with the attachment of Sch. pombe telomeric sequence. Previously, telomeric repeats from S. cerevisiae (Guerrini et al., 1985; Hahnenberger et al., 1989) and Tetrahymena (Sugawara and Szostak, 1986; Shervington et al., 1993) have been reported to form functional telomeres in Sch. pombe, though the efficiency of this event varied considerably between reports. Of 20 YACs transfected into Sch. pombe, only one was linear ( Hahnenberger et al., 1989). However, Guerrini et al. (1985) transfected linear plasmids terminating at both ends in S. cerevisiae telomeric sequence into Sch. pombe, and all of the analysed colonies contained linear episomes. The telomeres used in both experiments consisted of Tetrahymena telomeric sequence capped by TG1–3 sequence following introduction into S. cerevisiae. Similarly, Tetrahymena telomeric sequence on small linear plasmids was found to seed telomeres in Sch. pombe in all 24 clones analysed by Shervington et al.
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(1993), whilst Sugawara and Szostak (1986) found that only one in 11 transformants possessed an unrearranged linear molecule. Both studies used blocks of 300 bp of T G repeats at both ends, and the reason for these 2 4 differences is not obvious. The high efficiency of telomere formation by human and Tetrahymena sequences described here mirrors the efficiencies reported by Guerrini et al. (1985) and Shervington et al. (1993). Thus, it appears that Sch. pombe, like S. cerevisiae, is able to efficiently seed telomere formation from a variety of telomeric sequences. The loss of a substantial proportion of the 800 bp human telomere sequence during the addition of between 100 and 250 bp of Sch. pombe sequence in the clone analysed here is a process similar to that described in S. cerevisiae (Cross et al., 1990). The neo-telomeres here were also shown to be heterogeneous, exhibiting both interclonal and intraclonal variation. An analysis of yeast telomeres by Shampay and Blackburn (1988) revealed that the mean telomere size in a clonal population depends on the telomere length in the original clone (Shampay and Blackburn, 1988). Extrapolating this to Sch. pombe, the clone exhibiting an average size of 500 bp probably suffered less degradation before the addition of Sch. pombe telomeres than the clone exhibiting a mean size of 300 bp. Shampay and Blackburn (1988) also demonstrated that a similar range of heterogeneous telomere lengths develops in clones regardless of their initial size. This was seen here as all clones exhibited a telomere heterogeneity ranging from between 150 and 200 bp. The observation that a human telomere array is able to efficiently seed telomere formation in Sch. pombe indicates that a MAC could potentially be shuttled from a mammalian cell into Sch. pombe, and it would be maintained as a linear molecule. This could be useful for the characterization of minichromosomes formed in mammalian cells. However, after propagation in Sch. pombe, the human telomere array is severely truncated during the addition of Sch. pombe telomere array. In S. cerevisiae, similar composite telomeres have been shown to be rather inefficient at seeding telomeres after transfer into mammalian cells ( Taylor et al., 1994), which have a very strict sequence requirement for telomere formation (Hanish et al., 1994). Therefore, it will probably still be necessary to retrofit a MAC maintained in Sch. pombe with extra human telomere sequences prior to transfer into mammalian cells, as has been done for constructs propagated in S. cerevisiae (Ikeno et al., 1998). In order to determine whether Sch. pombe is a good host for cloning alphoid DNA, we used two BACs carrying 130 and 173 kb of alphoid DNA that have previously been shown to be able to form a centromere when transfected into HT1080 cells (Harrington et al., 1997). These BACs were retrofitted with a Sch. pombe
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ars1 and ura4+ selectable marker using an in-vitro recombination between loxP sites catalysed by Cre protein. Following electroporation into E. coli, all the clones analysed were correctly retrofitted with the ars1 and ura4+ gene, and most of the clones were unrearranged. Thus, this method of Cre-lox retrofitting is very efficient and should be of general use in adding DNA to BAC clones. In five of the 14 alphoid clones, the alphoid DNA had deleted, indicating that alphoid DNA is not completely stable when introduced into E. coli. Furthermore, as two of the three 173 kb alphoid BACs were rearranged, this length of DNA may be approaching the upper size limit for cloning alphoid DNA in E. coli. After transfection into Sch. pombe, the alphoid DNA had deleted down to less than 40 kb in all 14 clones analysed, whereas two control BACs containing complex human DNA had not deleted in nine out of 10 clones. This clearly indicates that alphoid DNA is very unstable when transfected into this Sch. pombe host. This is comparable with the widely reported instability of centromeric arrays in S. cerevisiae. Analysis of YAC libraries constructed in both recombination-proficient ( RAD+) and recombination-deficient (rad) S. cerevisiae strains showed a significant underrepresentation of YACs containing repetitive DNA (McGuigan et al., 1998). These YACs are smaller than expected and often demonstrate instability upon growth, generating differently sized molecules from a single clone. However, YACs carrying 140 and 190 kb of pure satellite DNA have been isolated from the recombination-deficient strains, rad52 (McGuigan et al., 1998) and rad54 (Le and Dobson, 1997), respectively, and even in RAD+ strains YACs containing pure satellite DNA stabilise at approximately 100 kb (McGuigan et al., 1998). Therefore, the rearrangement of alphoid DNA observed here in Sch. pombe appears extreme by comparison and would be expected to be a problem in SPARC libraries of mammalian DNA, possibly leading to underrepresentation and deletion of repetitive DNA. In S. cerevisiae, the observed differences in stability of satellite-containing YACs in rad and RAD+ strains demonstrate the role of recombination in the deletion and rearrangement of these YACs. It is probable that Sch. pombe’s recombinational mechanisms also account for the alphoid instability observed here. As both the FY562 strain and the rad52 S. cerevisiae strain exhibit a reduction in intragenic mitotic recombination, the FY562 strain used here should offer some protection against intramolecular recombination. A completely recombination-deficient Sch. pombe host may alleviate the problem of instability. The BAC-based molecules lack Sch. pombe centromeric sequences necessary for single copy maintenance, so there is the opportunity for intermolecular recombination between the multiple molecules in each cell. The
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addition of sequences conferring single copy maintenance to Sch. pombe vectors might increase the stability of the alphoid DNA. Functional analysis of minimal sequences conferring centromeric function have shown that at least 25 kb of sequence from cen1 is required for partial mitotic centromere function, and that this figure must be increased to 67 kb to approach the stability of the intact cen1 (Hahnenberger et al., 1991). Therefore, a centromere would result in an unfeasibly large cloning vector unsuitable for library construction. It is also possible that the marked instability may be due to the lack of intrinsic ars activity in alphoid DNA; only the single ars1 present in the vector would drive replication of the whole molecule, possibly imposing a maximum size of about 50 kb. Therefore, it may not be possible to clone large arrays of alphoid DNA in Sch. pombe without additional ars sequences. The larger size of Sch. pombe’s own chromosomes and the stability of its own repetitive DNA have led to the fission yeast being propounded as an alternative to S. cerevisiae for the cloning of mammalian, genomic DNA. However, overall, our observations indicate that Sch. pombe is not a useful host for propagating mammalian artificial chromosomes and may have no advantage over S. cerevisiae in the generation of future genomic libraries.
Acknowledgements We would like to thank Dr. Robin Allshire for supplying Sch. pombe strains, plasmids and advice, Dr. John Harrington and Dr Bruce Birren for providing the BACs, Dr. Paul Sadowski and Linda Beatty for the gift of Cre recombinase and Dr. Nik Davies and Nicola Light for help in initial steps in this work. This work was supported by MRC grant PG9227439, and K.M. was supported by an MRC studentship.
References Allshire, R., 1992. Manipulation of large minichromosomes in Schizosaccharomyces pombe with liposome-enhanced transformation. Meth. Enzymol. 216, 614–631. Allshire, R.C., Cranston, G., Gosden, J.R., Maule, J.C., Hastie, N.D., Fantes, P.A., 1987. A fission yeast chromosome can replicate autonomously in mouse cells. Cell 50, 391–403. Allshire, R.C., Dempster, M., Hastie, N.D., 1989. Human telomeres contain at least three types of G-rich repeat distributed non-randomly. Nucleic Acids Res. 17, 4611–4627. Buerstedde, J.M., Takeda, S., 1991. Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67, 179–188. Cross, S., Lindsey, J., Fantes, J., McKay, S., McGill, N., Cooke, H., 1990. The structure of a subterminal repeated sequence present on many human chromosomes. Nucleic Acids Res. 18, 6649–6657. Dunham, I., Lengauer, C., Cremer, T., Featherstone, T., 1992. Rapid
generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction. Hum. Genet. 88, 457–462. Guerrini, A.M., Ascenzioni, F., Tribioli, C., Donini, P., 1985. Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2m sequences. EMBO J. 4, 1569–1573. Gysler-Junker, A., Bodi, Z., Kohli, J., 1991. Isolation and characterization of Schizosaccharomyces pombe mutants affected in mitotic recombination. Genetics 128, 495–504. Hahnenberger, K.M., Baum, M.P., Polizzi, C.M., Carbon, J., Clarke, L., 1989. Construction of functional artificial minichromosomes in the fission yeast Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. USA 86, 577–581. Hahnenberger, K.M., Carbon, J., Clarke, L., 1991. Identification of DNA regions required for mitotic and meiotic functions within the centromere of Schizosaccharomyes pombe chromosome I. Mol. Cell. Biol. 11, 2206–2215. Halverson, D., Baum, M., Stryker, J., Carbon, J., Clarke, L., 1997. A centromere DNA-binding protein from fission yeast affects chromosome segregation and has homology to human CENP-B. J. Cell Biol. 136, 487–500. Hanish, J.P., Yanowitz, J.L., DeLange, T., 1994. Stringent sequence requirements for the formation of human telomeres. Proc. Natl. Acad. Sci. USA 91, 8861–8865. Harrington, J.J., Van Bokkelen, G., Mays, R.W., Gustashaw, K., Willard, H.F., 1997. Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nat. Genet. 15, 345–355. Heinzel, S.S., Krysan, P.J., Tran, C.T., Calos, M.P., 1991. Autonomous DNA replication in human cells is affected by the size and source of the DNA. Mol. Cell. Biol. 11, 2263–2272. Henning, K.A., Novotny, E.A., Compton, S.T., Guan, X.-Y., Lui, P.P., Ashlock, M.A., 1999. Human artificial chromosomes generated by modification of a yeast artificial chromosome containing both human alpha satellite sequences and single-copy DNA sequences. Proc. Natl. Acad. Sci. 96, 592–597. Ikeno, M., Grimes, B., Okazaki, T., Nakano, M., Saitoh, K., Hoshino, H., McGill, N.I., Cooke, H., Masumoto, H., 1998. Construction of YAC-based mammalian artificial chromosomes. Nat. Biotech. 16, 431–436. Kim, S.Y., Horrigan, S.K., Altenhofen, J.L., Arbieva, Z.H., Hoffman, R., Westbrook, C.A., 1998. Modification of bacterial artficial chromosome clones using cre recombinase: introduction of selectable markers for expression in eukaryotic cells. Genome Res. 8, 404–412. Kouprina, N., Eldarov, M., Moyzis, R., Resnick, M., Larionov, V., 1994. A model system to assess the integrity of mammalian YACs during transformation and propagation in yeast. Genomics 21, 7–17. Le, Y., Dobson, M.J., 1997. Stabilization of yeast artificial chromosome clones in a rad54-3 recombination-deficient host strain. Nucleic Acids Res. 25, 1248–1253. Lee, J.K., Huberman, J.A., Hurwitz, J., 1997. Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. USA 94, 8427–8432. McGuigan, A., Huxley, C., 1996. Replication of yeast DNA and novel chromosome formation in mouse cells. Nucleic Acids Res. 24, 2271–2280. McGuigan, A., Manson, A., Haldane, M., Huxley, C., 1998. Comparison of YACs containing mouse centromeric satellite sequences cloned in rad52 and RAD52 host strains. Mamm. Genome 9, 312–315. Mills, W., Critcher, R., Lee, C., Farr, C.J., 1999. Generation of an #2.4 Mb human X centromere-based minichromosome by targeted telomere-associated chromosome fragmentation in DT40. Hum. Mol. Genet. 8, 751–761. Neil, D.L., Villasante, A., Fisher, R.B., Vetrie, D., Cox, B., Tyler-
K.L. Mann, C. Huxley / Gene 241 (2000) 275–285 Smith, C., 1990. Structural instability of human tandemly repeated DNA sequences in yeast artificial chromosome vectors. Nucleic Acids Res. 18, 1421–1428. Nimmo, E.R., Cranston, G., Allshire, R.C., 1994. Telomere-associated chromosome breakage in fission yeast results in variegated expression of adjacent genes. EMBO J. 13, 3801–3811. Pluta, A.F., Zakian, V.A., 1989. Recombination occurs during telomere formation in yeast. Nature 337, 429–433. Shaikh, A.C., Sadowski, P.D., 1997. The Cre recombinase cleaves the lox site in trans. J. Biol. Chem. 272, 5695–5702. Shampay, J., Blackburn, E.H., 1988. Generation of telomere-length heterogeneity in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 85, 534–538. Shen, M.H., Yang, J., Loupart, M.L., Smith, A., Brown, W., 1997. Human mini-chromosomes in mouse embryonal stem cells. Hum. Mol. Genet. 6, 1375–1382. Shervington, A.A., Sparey, J.A., Bostok, C.J., 1993. Behaviour of plas-
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mid contaiing C4A2 repeats in S. cerevisiae and S. pombe. Biochem. Mol. Biol. Int. 29, 673–685. Steiner, N.C., Hahnenberger, K.M., Clarke, L., 1993. Centromeres of the fission yeast Schizosaccharomyces pombe are highly variable genetic loci. Mol. Cell. Biol. 13, 4578–4587. Sugawara, N., Szostak, J.W., 1986. Telomeres of Schizosaccharomyces pombe. Yeast 2 Suppl., S373. Sugawara, N., 1989. DNA sequences at the telomeres of the fission yeast S. pombe. PhD Thesis, Harvard University, Cambridge, MA. Taylor, S.S., Larin, Z., Tyler Smith, C., 1994. Addition of functional human telomeres to YACs. Hum. Mol. Genet. 3, 1383–1386. Tolmachova, T., Simpson, K., Huxley, C., 1999. Analysis of a YAC with human telomeres and oriP from Epstein–Barr virus in yeast and 293 cells. Nucleic Acids Res. 27, 3726–3744. Young, D.J.D., Nimmo, E.R., Allshire, R.C., 1998. A Schizosaccharomyces pombe artificial chromosome large DNA cloning system. Nucleic Acids Res. 26, 5052–5060.
Investigation of Schizosaccharomyces pombe as a cloning host for human telomere and alphoid DNA Kathryn L. Mann, Clare Huxley * Section of Molecular Genetics, Division of Biomedical Sciences, Imperial College School of Medicine, Sir Alexander Fleming Building, London SW7 2AZ, UK Received 23 August 1999; received in revised form 17 October 1999; accepted 29 October 1999 Received by V. Larionov
Abstract The fission yeast Schizosaccharomyces pombe (Sch. pombe) has been proposed as a possible cloning host for both mammalian artificial chromosomes (MACs) and mammalian genomic libraries, due to the large size of its chromosomes and its similarity to higher eukaryotic cells. Here, it was investigated for its ability to form telomeres from human telomere sequence and to stably maintain long stretches of alphoid DNA. Using linear constructs terminating in the telomere repeat, T AG , human telomere 2 3 DNA was shown to efficiently seed telomere formation in Sch. pombe. Much of the human telomeric sequence was removed on addition of Sch. pombe telomeric sequence, a process similar to that described in S. cerevisiae. To investigate the stability of alphoid DNA in fission yeast, bacterial artificial chromosomes (BACs) containing 130 and 173 kb of alphoid DNA were retrofitted with the Sch. pombe ars1 element and ura4+ marker using Cre-lox recombination. These alphoid BACs were found to be highly unstable in Sch. pombe deleting down to less than 40 kb, whilst control BACs of 96 and 202 kb, containing non-repetitive DNA, were unrearranged. Alphoid DNA has been shown to be sufficient for human centromere function, and this marked instability excludes Sch. pombe as a useful cloning host for mammalian artificial chromosomes. In addition, regions containing repetitive DNA from mammalian genomes may not be truly represented in libraries constructed in Sch. pombe. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Bacterial artificial chromosome; Cre recombinase; Mammalian artificial chromosome; Schizosaccharomyces pombe artificial chromosome library
1. Introduction The field of mammalian artificial chromosome (MAC ) research has recently been stimulated by the generation of minichromosomes by truncation of preexisting chromosomes or by transfection of alphoid DNA into human cells (Harrington et al., 1997; Shen et al., 1997; Ikeno et al., 1998; Mills et al., 1999). The generation of a MAC provides not only an immediate opportunity to study the elements required for chromosome structure and function but also the possibility of Abbreviations: ars, autonomously replicating sequence; BAC, bacterial artificial chromosome; Cm, chloramphenicol; Km, kanamycin; MAC, mammalian artificial chromosome; NR, DNA non-resolving DNA; PFG, pulsed-field gel; SPARC, Schizosaccharomyces pombe artificial chromosome; TAS, telomere associated repeats. * Corresponding author. Tel.: +44-207-594-3028; fax: +44-207-594-3015. E-mail address: [email protected] (C. Huxley)
developing new vectors for gene therapy and for making transgenic animals. A stable, segregating, linear chromosome requires three functional components; centromeres, telomeres and origins of replication. Alphoid DNA has been shown to be sufficient for centromere formation after transfection into HT1080 cells ( Harrington et al., 1997; Ikeno et al., 1998; Henning et al., 1999), telomere formation requires hundreds of bp of the telomere repeat T AG (Hanish et al., 1994), and 2 3 large fragments of DNA are able to replicate extrachromosomally in mammalian cells (Heinzel et al., 1991; McGuigan and Huxley, 1996). A useful MAC cloning host should allow the propagation of the intact MAC, modification of the MAC (probably by efficient homologous recombination) and transfer of the MAC into other cells where it would be maintained in an unrearranged form. E. coli has been used to clone the alphoid DNA arrays used for minichromosome formation by transfec-
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tion into HT1080 cells ( Harrington et al., 1997). The resulting minichromosomes were far larger than the input DNA, which must have recombined during chromosome formation. This experiment demonstrated that alphoid DNA is stable in E. coli with arrays of 173 kb of alpha satellite in BAC vectors being maintained upon extended growth. However, E. coli currently has a cloning capacity of up to 300 kb, which is limited by the inability to introduce larger DNA molecules into cells intact. In contrast, the smallest mammalian chromosomes are still 2.4 Mb in size (Mills et al., 1999). S. cerevisiae has also been used to clone alphoid DNA prior to transfection into HT1080 cells (Ikeno et al., 1998; Henning et al., 1999). Again, the input DNA was rearranged during minichromosome formation. S. cerevisiae’s main drawback as a MAC cloning host is that arrays of centromeric satellite DNA much larger than 100 kb are unstable (Neil et al., 1990; McGuigan et al., 1998). This is due to the efficient recombination of the tandemly repetitive DNA into smaller arrays. Alternative yeast strains deficient in genes involved in recombination, including rad52, have proved to be more stable cloning hosts for repetitive DNA, although the arrays cloned in these hosts are still relatively small (Neil et al., 1990; Ikeno et al., 1998; McGuigan et al., 1998). A more promising cloning host is the chicken DT40 cell line. Chickens and mammals share telomeric sequences, mammalian chromosomes segregate accurately in DT40 cells (Shen et al., 1997), and, importantly, this cell line shows high rates of recombination between chromosomal DNA and homologous exogenous DNA (Buerstedde and Takeda, 1991). A limitation is that the techniques that have been developed to transfer mini chromosomes between DT40 and other cell lines, such as microcell fusion, are inefficient and technically difficult. Purification of the MAC construct from DT40 cells will always be inefficient as they are megabase molecules maintained at single copy, and the host genome is large. Finally, Sch. pombe, a fission yeast, is a potential MAC cloning host. Its endogenous chromosomes possess several promising features compared to those of S. cerevisiae. Its largest chromosome is 5.7 Mb (as opposed to about 1.6 Mb in S. cerevisiae), indicating that it could accommodate MAC-sized DNA. Its centromeres are also more similar to those of higher eukaryotes, consisting of 35–110 kb of untranscribed, repetitive DNA with the repetitive elements arranged in both inverted and tandem formations around a non-repetitive central core (Steiner et al., 1993). Furthermore, Sch. pombe’s telomere associated sequence ( TAS ) exhibits a satellitelike arrangement with 86–89 bp repeating units forming higher order repeats of 0.9–1.2 kb, which in turn form arrays totalling at least 19 kb in length (Sugawara, 1989). These features suggest that the stability of repeti-
tive DNA may be greater in fission yeast than in S. cerevisiae. Sch. pombe artificial chromosome (SPARC ) vectors have been developed, containing Sch. pombe telomeric sequences ( T AC A C G ) (Sugawara and 2 0–1 0–1 0–1 1–8 Szostak, 1986), an autonomously replicating sequence (ars1) for replication and selectable markers (Nimmo et al., 1994). These vectors have been used to successfully clone human DNA in Sch. pombe in a linear form ( Young et al., 1998); 50 kb cosmids were subcloned without rearrangements or circularization. Preliminary experiments to investigate the possibility of cloning NotI-digested genomic DNA generated clones containing SPARCs of between 80 and 450 kb with a copy number of between five and 10 copies per cell. These molecules were found to be relatively stable upon extended growth ( Young et al., 1998), indicating that this system could be used to generate genomic libraries of mammalian DNA. However, the stability of repetitive DNA in Sch. pombe was not investigated. The ability of human telomeric DNA to seed telomeres in Sch. pombe would allow a MAC to be maintained in a linear form when transferred from mammalian cells to Sch. pombe and would possibly allow it to be shuttled back into mammalian cells directly. Transfer of a modified 3.5 Mb Sch. pombe chromosome into a mouse cell line using cell-to-cell fusion has previously been demonstrated (Allshire et al., 1987). Both Tetrahymena (Sugawara and Szostak, 1986; Shervington et al., 1993) and S. cerevisiae (Guerrini et al., 1985; Hahnenberger et al., 1989) telomeric repeats have been shown to seed telomeres in Sch. pombe, though the efficiencies of telomere formation vary considerably, from 5 to 100%, between studies. Here, we investigate the capacity of human telomere sequence to seed telomere formation in Sch. pombe and the stability of alphoid DNA in Sch. pombe.
2. Materials and methods 2.1. Schizosaccharomyces pombe host strain The Sch. pombe strain FY562 (h+leu1-32ura4D18rec36-55) was used in all experiments. This strain exhibits a 10-fold reduction in intragenic mitotic recombination compared to the wild-type strain (GyslerJunker et al., 1991). In S. cerevisiae, tandemly repeated DNA such as Y chromosome alpha satellite has been shown to be more stable in the recombination-deficient rad52 strain (Neil et al., 1990; McGuigan et al., 1998). The rad52 strain is also deficient in intragenic recombination ( Kouprina et al., 1994), and thus it was thought that the FY562 strain might provide a more stable cloning environment for alphoid DNA than wild-type Sch. pombe.
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2.2. Plasmids and cloning The plasmids pTF2 (Tolmachova et al., 1999) containing 800 bp of human telomeric sequence, and pEN51 (Nimmo et al., 1994), containing 258 bp of Sch. pombe telomere repeats and 800 bp of TAS, were used in the telomere study. DNA was gel-purified using the Qiaex II gel purification kit (Qiagen). DNA ligase, calf intestinal alkaline phosphatase and all restriction enzymes were obtained from Gibco BRL. To construct the pTF2:pEN51 molecule, pEN51 was digested with SacI, dephosphorylated and digested with XhoI to give a 6.6 kb fragment. pTF2 was linearized with NotI, dephosphorylated and digested with SalI to give an 8.9 kb fragment with a complementary site to XhoI. Following gel purification, the fragments were ligated, and the correct 15.5 kb molecule (pTF2: pEN51) was isolated from non-ligated pTF2 and pEN51 arms, and from pTF2:pTF2 and pEN51:pEN51 ligation products by gel purification. The BAC retrofitting plasmid, pBLTK.ars1.ura4+, was made in the following way. pBT was made by cloning a 2.4 kb XmnI fragment from pBR322 (New England Biolabs) carrying the tetracycline resistance gene (Tcr) into the HincII site of pBluescript II SK (Stratagene). A 0.8 kb SmaI/HindIII fragment from pBS226 (Gibco BRL) containing the loxP site was cloned into the SmaI/HindIII sites of pBT to form pBLT. A 1.2 kb SacI fragment from pEN51 carrying the kanamycin resistance gene (kmr) was then inserted into the SacI sites of pBLT to give pBLTK. Finally, a 3.6 kb PvuII/XmnI fragment from pEN51 containing the Sch. pombe ars1 and ura4+ marker was cloned into the EcoRV site in the Tcr gene of pBLTK, to give pBLTK.ars1.ura4+. 2.3. Analysis of termini pTF2:pEN51 DNA was isolated from a Seaplaque low melting point agarose ( FMC ) pulsed field gel using beta-Agarase I (New England Biolabs) and recovered by ethanol precipitation. This DNA was used in the BAL-31 nuclease analysis. The DNA was digested with 0.01, 0.1, 0.3, 0.6, 1.0 and 3.0 U of BAL-31 for 20 min, at which point, the reaction was terminated by the addition of EGTA. The DNA was extracted with phenol/chloroform, precipitated with ethanol and digested with BglII. To clone the terminus prior to sequencing, the 3∞ G-rich telomere overhang was removed with varying amounts of BAL-31 nuclease (New England Biolabs) applied for 30 min at 30°C. The reaction was terminated with EGTA, and the DNA was reprecipitated and bluntended with T4 DNA polymerase (New England Biolabs). A terminal fragment was generated by KpnI digestion and cloned into SmaI/KpnI-cut pBluescript.
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Colony hybridization was used to identify clones carrying a human telomere insert. Colonies were inoculated in 96 well plates and transferred to Hybond-N filters placed on LB plates containing ampicillin using a microplate replicator. These were incubated at 37°C overnight. The filters were then prepared by standard methods and hybridized as described below with a human telomere probe. Positive colonies were sequenced using the M13F and M13R primers (Stratagene) and Amersham Life Sciences Sequenase Version 2.0 kit, following the manufacturer’s instructions. 2.4. Sch. pombe transfections, screening and DNA preparation The protocol described by Allshire (1992) was followed except for the replacement of the spheroplasting enzyme NovoZym 234 by Zymolyase 100T (ICN ). This enzyme is partially purified by affinity chromatography, contains no RNase or DNase activity and consistently results in higher transfection and faster colony growth. Between 1.5 and 2.5×108 cells were incubated with 6 mg of Zymolyase 100T at 37°C until 70–90% of cells had spheroplasted. For colony hybridization, Sch. pombe colonies were streaked on the appropriate plates and incubated at 30°C for 3 days. Hybond N filters (Amersham) were placed on the plates, which were incubated for a further 18 h. The filters were then placed in SP2 (1.2 M sorbitol, 50 mM sodium citrate, 50 mM sodium phosphate, pH 5.6) (Allshire, 1992)/b-mercaptoethanol (1 ml/ml SP2) for 15 min, followed by an overnight incubation at 37°C in SP2/b-mercaptoethanol/Zymolyase 100T (200 mg/ml ). Once 70–90% of cells had spheroplasted, the filters were treated, as described in Amersham’s instructions, and hybridized with the neor gene probe, as described below. Agarose plugs were prepared for the isolation of intact DNA. Cells were spheroplasted (Allshire, 1992), resuspended at a concentration of 2×109 cells/ml and mixed with an equal volume of 2% LMP agarose solution. This suspension was aliquoted into pre-chilled plug moulds and allowed to set. The plugs were incubated in a 1% LDS solution (1% dodecyl lithium sulphate, 100 mM EDTA, 10 mM Tris–Cl, pH 8.0) at 37°C for 1 h with gentle shaking, followed by a further incubation in 1% LDS, overnight at 37°C. The plugs were then washed twice for 2 h in 20% NDS (0.2% Nlauryl sarcosine, 100 mM EDTA, 2 mM Tris, pH 9.0) at room temperature and stored in this solution at 4°C. 2.5. BAC clones and DNA preparation The alphoid BACs, pBAC17a48 and pBAC17a64 were a gift of J. Harrington and contain 129.6 and 172.8 kb of pure alphoid DNA from chromosome 17,
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respectively (Harrington et al., 1997). The control BACs, L269 and L281, were a gift of B. Birren and are 96 and 202 kb, respectively. They were derived from chromosome 17, and sequence information is available from the Whitehead Institute/MIT Genome Sequencing Project. BAC DNA was prepared using a standard alkaline lysis miniprep method including a phenol/chloroform extraction.
telomere probe was a 0.8 kb NotI/SstI fragment from pSXneo-0.8-T AG (Hanish et al., 1994). Alphoid con2 3 sensus probe was amplified from total human genomic DNA using the a27 and a30 primers (Dunham et al., 1992). Finally, DNA from Caco 2 cells (ATCC ) was used as the total human DNA probe. These probes were labelled using the Megaprime DNA labelling system (Amersham), and unincorporated nucleotides were removed using NucTrap purification columns (Stratagene).
2.6. Retrofitting of BAC clones using the Cre-lox system Cre recombinase was a kind gift from P. Sadowski (Shaikh and Sadowski, 1997). Plasmid DNA was mixed with BAC DNA at equal molar concentrations and a total mass of between 100 and 500 ng. Ten times Cre reaction buffer (Novagen), 1 ml of Cre recombinase (3.6 mg/ml ) and H O were added to give a total reaction 2 volume of 30 ml which was incubated at 37°C for 1 h. The reaction was terminated by a phenol/chloroform extraction. DNA was recovered by ethanol precipitation and resuspended in 10 ml of H O. Two microlitres were 2 electroporated into 20 ml aliquots of ElectroMAX DH10B cells (Gibco BRL) at 1.4 kV, 100 V, 25 mF using a Bio-Rad Gene Pulser. 2.7. DNA analyses Agarose plugs of high-molecular-weight DNA were washed twice for 2 h in TE buffer at room temperature to remove the NDS. They were then equilibrated in the appropriate enzyme buffer for 2×30 min. The plugs were digested in 1× restriction buffer with 100 mg/ml of bovine serum albumin and between 40 and 60 U of restriction enzyme, overnight, at the recommended temperature. Between 0.5 and 1 mg of BAC DNA was digested for 2 h with 5–10 U of enzyme at the appropriate temperature. Pulsed field gel electrophoresis (PFGE ) was performed on a BIO-RAD CHEF-DRIII system. DNA was separated on 1% agarose gels, run in 0.5× TBE, and then transferred to Hybond N+ membrane (Amersham), as described by the manufacturer. Both the prehybridization and hybridization steps were carried out in Church buffer (16.8 g/l of NaH PO .H O, 2 4 2 54.1 g/l of Na HPO .12H O, 7% SDS, 100 mg/ml 2 4 2 salmon sperm DNA) at 65°C. Filters were washed in 2–0.5× SSC (20×: 175.3 g/l of NaCl, 88.2 g/l of sodium citrate, pH 7.0)/0.1% SDS at 65°C. The neor gene probe was a 1.1 kb XhoI/SalI fragment obtained from pMC1NeoPolyA (Strategene) The ura4+ gene probe (1.8 kb) and the ampr gene probe (2.1 kb) were isolated from pEN51 using a PvuII/ClaI digest. The LYS2 gene was a 5 kb EcoRI/HindIII fragment from pCH37, a plasmid derived from YCp50 with the LYS2 gene from YIp333 cloned into it. The human
3. Results 3.1. Human telomere function in Sch. pombe A linear molecule, pTF2:pEN51, was constructed from the pTF2 and pEN51 plasmids, to terminate at one end in 258 bp of Sch. pombe telomeric sequence and at the other with 800 bp of human telomeric sequences ( Fig. 1). This molecule was used to assess the telomere seeding efficiency of human telomeric sequence in Sch. pombe. To verify the human telomere sequence in pTF2, the 0.8 kb NotI/KpnI telomere fragment from the original telomere plasmid, pSXneo-0.8-T AG (Hanish et al., 2 3 1994), was subcloned and sequenced (Fig. 2). The sequence was unexpectedly found to be a complex mixture of human T AG and T G telomeric sequences, 2 3 2 4 with T AG arrays contributing 62% of the 654 bp 2 3 sequenced. A reasonable explanation for this is that the complex array may have been present in the human telomere from which this fragment was cloned. Mixed arrays of T AG , T G and TGAG sequences have 2 3 2 4 3 previously been found close to the terminal blocks of T AG in some human chromosomes (Allshire et al., 2 3 1989). This 800 bp sequence does seed efficient formation of telomeres in mammalian cells (Hanish et al., 1994; Tolmachova et al., 1999). The linear pTF2:pEN51 DNA molecules were transfected into Sch. pombe, and the pEN51 arm was selected for on ura− plates. Colony hybridization using a neor probe identified clones carrying the pTF2 arm.
Fig. 1. Map showing the linear pTF2:pEN51 construct. The molecule terminates in 258 bp of Sch. pombe telomeric sequence and 800 bp of human telomeric sequence and contains a ura4+ gene for selection purposes and an ars1 element for replication in Sch. pombe.
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Fig. 2. Partial sequence of the 0.8 kb human telomere fragment of pSXneo-0.8-T AG . The KpnI/NotI fragment was subcloned into pBluescript 2 3 and then sequenced using M13F and M13R primers. The M13F primer produced 222 bp of sequence from the KpnI end of the telomere. The M13R primer produced 414 bp of sequence from the NotI end of the human telomere fragment, representing the sequence at the very terminus of the pTF2:pEN51 molecule. Human T AG repeats are in bold. T G and other repeats are in normal type. 2 3 2 4
Ten clones were chosen for further analysis; six clones showing strong hybridization to the neor probe (clones 1–6), one control clone containing the original pEN51 plasmid (clone 7) and three clones showing no hybridization to the neor probe (clones 8–10). Pulsed field gel analysis and Southern blotting revealed that all the clones contained extrachromosomal elements (Fig. 3A). Clones 1–6, 8 and 10 exhibited bands corresponding to the linear 15.5 kb pTF2:pEN51 molecule when hybridized with neor, ura4+ and human telomere probes. Clones 8 and 10 were presumably false negatives from the colony hybridization due to inconsistent hybridization of the neor probe. Clone 7 contained the 9.1 kb circular pEN51 molecule and hybridized only to the ura4+ probe; the supercoiled molecules run more slowly than linears on this PFG, and the intense hybridization at the well is due to trapped open circular molecules. Clone 9 contained a 12 kb molecule, which again hybridized only to the ura4+ probe and is probably a linear pEN51:pEN51 molecule. To confirm that the molecules were linear, clone 5 was analysed after treatment with BAL-31 nuclease, an exonuclease that degrades both 3∞ and 5∞ termini of DNA, and BglII ( Fig. 3B). Hybridization with a LYS2 probe identified the pTF2 terminus, which exhibits a progressive reduction in size with increasing amounts of BAL-31 nuclease, indicating that it is a linear molecule. Hybridization with an ampr probe identified both the pEN51 terminus, again decreasing in size, and a constant band representing an internal fragment undigested by BAL-31 nuclease (data not shown). The telomere seeded in Sch. pombe by the human telomeric sequence on pTF2 was sized using a KpnI digest and Southern analysis (data not shown) and found to be heterogeneous, both within and between clones. Each clone was found to have a similar range of telomere size heterogeneity of 150–200 bp. However, the
average telomere length varied between clones with clone 2 having 220–420 bp, clone 3 having 380–600 bp and clone 5 having 300–450 bp of telomere array. To identify the length of the human telomeric sequence remaining at the terminus and to investigate the addition of Sch. pombe’s own telomeric repeats, the human telomere end of clone 5 was subcloned into pBluescript and sequenced. Two clones containing different human:Sch. pombe sequence junctions were identified. In one, Sch. pombe telomeric repeats are directly attached to 186 bp of the human telomere sequence disrupting a T AG array to form a 2 3 T AG :T ACACG junction (Fig. 3C ). No mixing of 2 3 2 2 the Sch. pombe and human arrays was observed, and the junction itself does not disrupt either repeat. Therefore, 614 bp of the human telomere array were removed during the addition of Sch. pombe telomeric sequence. As the telomere array from the KpnI site is between 300 and 450 bp in this clone, between 100 and 250 bp of Sch. pombe sequence have been added to the remaining 186 bp of human telomere sequence to form a functional telomere. The other clone was found to have a T G :T ACACG junction (data not shown), 2 4 2 2 with the Sch. pombe telomere attached to 216 bp of the human telomeric sequence. As the pTF2:pEN51 molecule terminates at one end in Sch. pombe telomeric repeats, the possibility that these were involved in the telomere formation process at the human telomeric terminus, perhaps by homologous recombination, was investigated. In similar experiments in S. cerevisiae, it has been demonstrated that recombination between the terminal sequences does occur prior to capping with host telomeric sequence (Pluta and Zakian, 1989). A linear molecule was constructed, terminating at both ends in 800 bp of human telomere and carrying the Sch. pombe ura4+ marker and ars1. Following transfection into Sch. pombe and pulsed
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Fig. 3. Analysis of Sch. pombe clones carrying pTF2:pEN51 molecules. (A) High-molecular-weight DNA from each of the 10 clones and the original FY562 strain of Sch. pombe were separated on a pulsed field gel, with 2–5 s switching, for 15 h at 14°C. The gel was Southern-blotted and the filter hybridized with the ura4+ probe. All clones contain extrachromosomal elements: clones 1–6, 8 and 10 exhibit DNA bands running at about 15.5 kb (although the bands in clones 6 and 10 are only visible with a longer exposure); clone 7 contains a circular pEN51 molecule, whilst clone 9 shows a band running at about 12 kb. The positions of the wells, the non-resolving DNA (NR) and the size markers are indicated at the left of the gel. (B) Extrachromosomal pTF2:pEN51 molecule from clone 5 was treated with varying amounts of BAL-31 nuclease as shown above each lane. The DNA was subsequently digested with BglII and separated on an agarose gel, which was Southern-blotted and hybridized with a LYS2 probe to identify the 4.7 kb human telomere arm. The gradual reduction in size of this fragment with increasing amounts of BAL-31 nuclease identifies this as a terminus, and thus the pTF2:pEN51 molecule as linear. The positions of size markers and the sizes of fragments are indicated on the left. (C ) Termini of the pTF2:pEN51 molecules from clone 5 were cloned and sequenced. The sequence shown illustrates the junction between human (T AG ) and Sch. pombe ( T AC A C G ) telomeric repeats. The mixed T AG and T G repeats are part of the complex 2 3 2 0–1 0–1 0–1 1–8 2 3 2 4 pattern of the human telomere fragment.
field gel analysis, three of seven clones carrying both arms were found to be linear and of the correct size (data not shown). As this two-armed molecule was not purified prior to transfection, a range of ligated and non-ligated molecules would also have been introduced into Sch. pombe, probably accounting for the lower frequency of correctly sized molecules observed here. However, this experiment does confirm the efficient
seeding of telomeres in Sch. pombe by human telomeric sequence, probably independent of intramolecular recombination. 3.2. Retrofitting BACs in vitro using Cre-lox In order to investigate the stability of large arrays of alphoid DNA in Sch. pombe we used two BAC clones,
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pBAC17a48 and pBAC17a64, which contain 130 and 173 kb of alphoid, respectively (Harrington et al., 1997). Alphoid BACs, rather than YACs, were chosen to investigate the stability of large alphoid arrays in Sch. pombe as circular molecules do not require telomere formation for extrachromosomal maintenance, and alphoid arrays of over 100 kb in size are reasonably stable in bacteria. For extrachromosomal maintenance in Sch. pombe, the BACs first had to be retrofitted with a marker, in this case, ura4+, and with an ars1 element. Initial attempts to sub-clone the BAC insert into a new vector containing these necessary elements were unsuccessful. Therefore, an alternative strategy was designed, exploiting the loxP site present in all BAC vectors. Here, Cre recombinase promotes site specific recombination between the loxP site on the BAC vector and a loxP site on the retrofitting plasmid, resulting in the insertion of relevant DNA on to the BAC, as shown in Fig. 4. A similar procedure has recently been published ( Kim et al., 1998). Four BACs were retrofitted, pBAC17a48 and pBAC17a64 containing alphoid DNA, and L269 and L281 containing 96 and 202 kb of single-copy sequence from chromosome 17, respectively. After mixing the BAC DNA with the retrofitting construct and Cre protein, the DNA was electroporated into E. coli. Colonies were selected for dual resistance to chloramphenicol (Cm) and kanamycin ( Km), to select for the BAC and retrofitting molecule, respectively. As the BssHII religated fragment lacks an origin of replication, it is unable to exist episomally and can only be maintained by integrating into the BAC or bacterial genome. BACs were analysed by restriction digestion and pulsed field gel analysis (data not shown). All were found to have the retrofitting DNA correctly inserted into the BAC loxP site. All of the retrofitted control BAC clones that were analysed were found to be of the correct size, whilst three out of 11 of the 130 kb alphoid BACs and two out of three of the 173 kb alphoid BACs were found to have deleted down to a smaller size. 3.3. Alphoid DNA stability in Sch. pombe The retrofitted alphoid and control BACs were transfected into Sch. pombe, and colonies were selected on ura− plates. Clones were analysed by restriction digestion, pulsed field gel and Southern analysis (Fig. 5). From the transfections of the alphoid BACs, 10 pBAC17a48 and four pBAC17a64 clones were analysed, and in all of them, the alphoid DNA was found to have deleted down to between 3 and 40 kb in size. Fig. 5A shows the 10 pBAC17a48 clones that have all deleted to less than 50 kb. Furthermore, three of the alphoid clones exhibit more than one band. Fig. 5B shows three of the pBAC17a64 clones, where the 173 kb input alphoid has been reduced to between 10 and 40 kb. The
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Fig. 4. Illustration of the BAC retrofitting strategy. Construction of the BAC retrofitting plasmid, pBLTK.ars1.ura4+, is described in Section 2.2. It carries the ura4+ and ars1 elements, a loxP site and a kmr marker for selection in bacteria. In order for the retrofitted BAC to be retained at single copy, the retrofitting plasmid’s origin (ColE1) must be removed prior to the Cre-lox reaction. This was achieved with a BssHII digestion of pBLTK.ars1.ura4+, which separates the ori and ampr backbone from the multiple cloning site containing the relevant elements for insertion onto the BAC. The appropriate BssHII fragment was gel-purified, religated and used in the Cre-lox reaction with BAC miniprep DNA. Cre recombinase promotes site specific recombination between the two loxP sites, resulting in the insertion of the ars1, ura4+ and kmr elements onto the BAC. As the religated BssHII fragment lacks an origin of replication, it is unable to exist episomally and can only be maintained by integrating into the BAC or bacterial genome. Therefore, following transfection, clones carrying the retrofitted BAC were selected for using double Km/Cm selection. All analysed clones contained a retrofitted BAC.
control BACs, L269 and L281 were also transfected into Sch. pombe, and five L269 clones and four L281 clones were analysed ( Fig. 5B). All were stable with the exception of one of the 202 kb L281 BACs, which had deleted to approximately 100 kb. A slight growth advantage displayed by clones containing alphoid DNA compared to those containing non-alphoid DNA was noted. Furthermore, two Sch. pombe proteins with homology to the mammalian alphoid-binding protein, CENP-B, have been identified,
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Fig. 5. Analysis of alphoid BACs in Sch. pombe. Agarose plugs of DNA were prepared from ura4+ Sch. pombe clones and digested with SalI or NotI to remove the BAC vector. The BAC inserts were resolved on a pulsed field gel, with 5–20 s switching for 20 h at 14°C. The gels were Southern blotted and the filters hybridized with an alphoid probe or total human DNA. (A) pBAC17a48 molecules in 10 Sch. pombe clones have deleted to less than 50 kb in size. The lane labelled ‘a YAC’ contains a 140 kb YAC containing alphoid DNA. (B) pBAC17a64 molecules in the 3 Sch. pombe clones have deleted to between 10 and 35 kb in size in comparison to the original 180 kb BAC run in the lane labelled ‘c’. Three of the L281 clones are unrearranged and run at the same size as the original 200 kb BAC run in the lane labelled ‘c’, while one clone has deleted to about 100 kb. All five of the L269 clones are unrearranged and give the same three SalI fragments as the original BAC in the lane labelled ‘c’. The positions of the wells and the NR DNA and the sizes of fragments are indicated on the left of the gel.
Abp1p and Cbh (Halverson et al., 1997; Lee et al., 1997). As the binding sites of these two proteins have not been fully characterized, we investigated the possibility that these proteins are binding to the alphoid DNA to give the molecules a segregational advantage in Sch. pombe. A mitotic stability assay (Hahnenberger et al., 1989) was carried out on clones containing approximately 40 kb of alphoid and those containing non-alphoid DNA. No difference was observed between the rate of loss of these molecules per generation, with both types being less stable than the pEN51 plasmid. An alternative explanation for the growth difference is that the non-alphoid molecules are generally larger due to their being unrearranged in Sch. pombe. Consequently, replication of these molecules may take longer and cause a slight delay in the cell cycle.
4. Discussion The data presented here show that human telomeric sequence can efficiently seed telomere formation in Sch. pombe. Following transfection into Sch. pombe of a 15.5 kb linear molecule terminating at one end with human telomere sequence, all eight of the neo and ura4+ positive clones that were analysed were found to
be linear. The two human:Sch. pombe telomere sequence junctions found may have been generated from separate telomere seeding events in two molecules or dynamic changes in telomere length in just one molecule that had already been stabilized by the addition of Sch. pombe telomeres. It was also demonstrated that a substantial proportion of the 800 bp of human telomere sequence is removed concomitantly with the attachment of Sch. pombe telomeric sequence. Previously, telomeric repeats from S. cerevisiae (Guerrini et al., 1985; Hahnenberger et al., 1989) and Tetrahymena (Sugawara and Szostak, 1986; Shervington et al., 1993) have been reported to form functional telomeres in Sch. pombe, though the efficiency of this event varied considerably between reports. Of 20 YACs transfected into Sch. pombe, only one was linear ( Hahnenberger et al., 1989). However, Guerrini et al. (1985) transfected linear plasmids terminating at both ends in S. cerevisiae telomeric sequence into Sch. pombe, and all of the analysed colonies contained linear episomes. The telomeres used in both experiments consisted of Tetrahymena telomeric sequence capped by TG1–3 sequence following introduction into S. cerevisiae. Similarly, Tetrahymena telomeric sequence on small linear plasmids was found to seed telomeres in Sch. pombe in all 24 clones analysed by Shervington et al.
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(1993), whilst Sugawara and Szostak (1986) found that only one in 11 transformants possessed an unrearranged linear molecule. Both studies used blocks of 300 bp of T G repeats at both ends, and the reason for these 2 4 differences is not obvious. The high efficiency of telomere formation by human and Tetrahymena sequences described here mirrors the efficiencies reported by Guerrini et al. (1985) and Shervington et al. (1993). Thus, it appears that Sch. pombe, like S. cerevisiae, is able to efficiently seed telomere formation from a variety of telomeric sequences. The loss of a substantial proportion of the 800 bp human telomere sequence during the addition of between 100 and 250 bp of Sch. pombe sequence in the clone analysed here is a process similar to that described in S. cerevisiae (Cross et al., 1990). The neo-telomeres here were also shown to be heterogeneous, exhibiting both interclonal and intraclonal variation. An analysis of yeast telomeres by Shampay and Blackburn (1988) revealed that the mean telomere size in a clonal population depends on the telomere length in the original clone (Shampay and Blackburn, 1988). Extrapolating this to Sch. pombe, the clone exhibiting an average size of 500 bp probably suffered less degradation before the addition of Sch. pombe telomeres than the clone exhibiting a mean size of 300 bp. Shampay and Blackburn (1988) also demonstrated that a similar range of heterogeneous telomere lengths develops in clones regardless of their initial size. This was seen here as all clones exhibited a telomere heterogeneity ranging from between 150 and 200 bp. The observation that a human telomere array is able to efficiently seed telomere formation in Sch. pombe indicates that a MAC could potentially be shuttled from a mammalian cell into Sch. pombe, and it would be maintained as a linear molecule. This could be useful for the characterization of minichromosomes formed in mammalian cells. However, after propagation in Sch. pombe, the human telomere array is severely truncated during the addition of Sch. pombe telomere array. In S. cerevisiae, similar composite telomeres have been shown to be rather inefficient at seeding telomeres after transfer into mammalian cells ( Taylor et al., 1994), which have a very strict sequence requirement for telomere formation (Hanish et al., 1994). Therefore, it will probably still be necessary to retrofit a MAC maintained in Sch. pombe with extra human telomere sequences prior to transfer into mammalian cells, as has been done for constructs propagated in S. cerevisiae (Ikeno et al., 1998). In order to determine whether Sch. pombe is a good host for cloning alphoid DNA, we used two BACs carrying 130 and 173 kb of alphoid DNA that have previously been shown to be able to form a centromere when transfected into HT1080 cells (Harrington et al., 1997). These BACs were retrofitted with a Sch. pombe
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ars1 and ura4+ selectable marker using an in-vitro recombination between loxP sites catalysed by Cre protein. Following electroporation into E. coli, all the clones analysed were correctly retrofitted with the ars1 and ura4+ gene, and most of the clones were unrearranged. Thus, this method of Cre-lox retrofitting is very efficient and should be of general use in adding DNA to BAC clones. In five of the 14 alphoid clones, the alphoid DNA had deleted, indicating that alphoid DNA is not completely stable when introduced into E. coli. Furthermore, as two of the three 173 kb alphoid BACs were rearranged, this length of DNA may be approaching the upper size limit for cloning alphoid DNA in E. coli. After transfection into Sch. pombe, the alphoid DNA had deleted down to less than 40 kb in all 14 clones analysed, whereas two control BACs containing complex human DNA had not deleted in nine out of 10 clones. This clearly indicates that alphoid DNA is very unstable when transfected into this Sch. pombe host. This is comparable with the widely reported instability of centromeric arrays in S. cerevisiae. Analysis of YAC libraries constructed in both recombination-proficient ( RAD+) and recombination-deficient (rad) S. cerevisiae strains showed a significant underrepresentation of YACs containing repetitive DNA (McGuigan et al., 1998). These YACs are smaller than expected and often demonstrate instability upon growth, generating differently sized molecules from a single clone. However, YACs carrying 140 and 190 kb of pure satellite DNA have been isolated from the recombination-deficient strains, rad52 (McGuigan et al., 1998) and rad54 (Le and Dobson, 1997), respectively, and even in RAD+ strains YACs containing pure satellite DNA stabilise at approximately 100 kb (McGuigan et al., 1998). Therefore, the rearrangement of alphoid DNA observed here in Sch. pombe appears extreme by comparison and would be expected to be a problem in SPARC libraries of mammalian DNA, possibly leading to underrepresentation and deletion of repetitive DNA. In S. cerevisiae, the observed differences in stability of satellite-containing YACs in rad and RAD+ strains demonstrate the role of recombination in the deletion and rearrangement of these YACs. It is probable that Sch. pombe’s recombinational mechanisms also account for the alphoid instability observed here. As both the FY562 strain and the rad52 S. cerevisiae strain exhibit a reduction in intragenic mitotic recombination, the FY562 strain used here should offer some protection against intramolecular recombination. A completely recombination-deficient Sch. pombe host may alleviate the problem of instability. The BAC-based molecules lack Sch. pombe centromeric sequences necessary for single copy maintenance, so there is the opportunity for intermolecular recombination between the multiple molecules in each cell. The
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addition of sequences conferring single copy maintenance to Sch. pombe vectors might increase the stability of the alphoid DNA. Functional analysis of minimal sequences conferring centromeric function have shown that at least 25 kb of sequence from cen1 is required for partial mitotic centromere function, and that this figure must be increased to 67 kb to approach the stability of the intact cen1 (Hahnenberger et al., 1991). Therefore, a centromere would result in an unfeasibly large cloning vector unsuitable for library construction. It is also possible that the marked instability may be due to the lack of intrinsic ars activity in alphoid DNA; only the single ars1 present in the vector would drive replication of the whole molecule, possibly imposing a maximum size of about 50 kb. Therefore, it may not be possible to clone large arrays of alphoid DNA in Sch. pombe without additional ars sequences. The larger size of Sch. pombe’s own chromosomes and the stability of its own repetitive DNA have led to the fission yeast being propounded as an alternative to S. cerevisiae for the cloning of mammalian, genomic DNA. However, overall, our observations indicate that Sch. pombe is not a useful host for propagating mammalian artificial chromosomes and may have no advantage over S. cerevisiae in the generation of future genomic libraries.
Acknowledgements We would like to thank Dr. Robin Allshire for supplying Sch. pombe strains, plasmids and advice, Dr. John Harrington and Dr Bruce Birren for providing the BACs, Dr. Paul Sadowski and Linda Beatty for the gift of Cre recombinase and Dr. Nik Davies and Nicola Light for help in initial steps in this work. This work was supported by MRC grant PG9227439, and K.M. was supported by an MRC studentship.
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