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Human Molecular Genetics Pages 751-761  

Generation of an ~2.4 Mb human X centromere-based minichromosome by targeted telomere-associated chromosome fragmentation in DT40
Introduction
Results
   Generation of a DT40-human Xp microcell hybrid
   Targeting strategy for the generation of an X centromere-based human minichromosome
   Identification of homologous recombinants
   Cytogenetic analysis of the ZXDA targeted cell lines
   Molecular analysis of experimentally seeded telomeres
   Pulsed-field gel electrophoresis (PFGE) of the targeted chromosomes
   Molecular composition of the X-derived minichromosome present in IKNFA3
   Stability of the minichromosome in DT40 and after transfer to mammalian cell lines
Discussion
   Generation of a human minichromosome in DT40
   Molecular and cytogenetic examination of the linear FA3 minichromosome
   Mitotic stability of the FA3 minichromosome in DT40
   Mitotic stability of the FA3 minichromosome in established human and rodent cell lines
   Loss rates, copy number control and a lower size limit?
Materials And Methods
   Microcell-mediated chromosome transfer
   Recombinant DNA constructs
   Transfection of the human Xp-DT40 hybrid, IG4
   DNA preparation, digestion and analysis
   Probes
   PFGE
   In situ hybridization
   Preparation of mechanically stretched chromosomes and chromosome fibres
   Mitotic stabilities
Acknowledgements
References

Generation of an ~2.4 Mb human X centromere-based minichromosome by targeted telomere-associated chromosome fragmentation in DT40

Generation of an ~2.4 Mb human X centromere-based minichromosome by targeted telomere-associated chromosome fragmentation in DT40

Walter Mills, Ricky Critcher, Charles Lee1 and Christine J. Farr*

Departments of Genetics and 1Pathology, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK

Received January 7, 1999; Revised and Accepted February 22, 1999

A linear mammalian artificial chromosome (MAC) will require at least three types of functional element: a centromere, two telomeres and origins of replication. As yet, our understanding of these elements, as well as many other aspects of structure and organization which may be critical for a fully functional mammalian chromosome, remains poor. As a way of defining these various requirements, minichromosome reagents are being developed and analysed. Approaches for minichromosome generation fall into two broad categories: de novo assembly from candidate DNA sequences, or the fragmentation of an existing chromosome to reduce it to a minimal size. Here we describe the generation of a human minichromosome using the latter, top-down, approach. A human X chromosome, present in a DT40-human microcell hybrid, has been manipulated using homologous recombination and the targeted seeding of a de novo telomere. This strategy has generated a linear ~2.4 Mb human X centromere-based minichromosome capped by two artificially seeded telomeres: one immediately flanking the centromeric [alpha]-satellite DNA and the other targeted to the zinc finger gene ZXDA in Xp11.21. The chromosome retains an [alpha]-satellite domain of ~1.8 Mb, a small array of [gamma]-satellite repeat (~40 kb) and ~400 kb of Xp proximal DNA sequence. The mitotic stability of this minichromosome has been examined, both in DT40 and following transfer into hamster and human cell lines. In all three backgrounds, the minichromosome is retained efficiently, but in the human and hamster microcell hybrids its copy number is poorly regulated. This approach of engineering well-defined chromosome reagents will allow key questions in MAC development (such as whether a lower size limit exists) to be addressed. In addition, the 2.4 Mb minichromosome described here has potential to be developed as a vector for gene delivery.

INTRODUCTION

Information about the various functional elements of chromosomes in higher eukaryotes has come from a variety of sources, including experiments attempting to create the various structural elements de novo (1-5) and through the manipulation of existing chromosomes (6-11). Inevitably, experiments designed to extend our understanding of mammalian chromosomes are inextricably linked with attempts to generate artificial mammalian chromosome vectors which, as well as providing an alternative and potentially powerful research tool for both tissue culture- and transgenic-based experiments, might also, in the longer term, provide an alternative strategy for somatic gene therapy (12,13).

By far the best understood functional element of the mammalian chromosome, at least at the DNA level, is the telomere. Through characterization of both naturally occurring (14-16) and experimentally generated (1-3) chromosome truncation events, it has been established that the only DNA requirement for mitotic telomere function is a short tandem array of the repeat (TTAGGG)n. More recently, a similar approach has been undertaken to determine the DNA requirements for de novo centromere formation in human cells (4,5). These experiments strongly suggest that [alpha]-satellite DNA, which is present at all naturally occurring human centromeres, plays a crucial role in mitotic centromere function. However, in light of the complex nature of the products generated in these experiments and the recent identification of some neo-centromeres lacking [alpha]-satellite DNA (17,18), the DNA requirements of human centromeres have yet to be fully elucidated. Currently, our understanding of the DNA sequence requirements (if any) for the initiation of DNA synthesis is limited (19,20). However, since it is generally assumed that DNA capable of providing origin function will be present in most large pieces of DNA, current efforts to build an artificial chromosome vector have focused on the requirements for centromere function (21).

We previously have reported the generation of an ~8 Mb human minichromosome by the fragmentation of an existing chromosome (8). The approach relied on random integration of telomere seeding constructs into a non-essential human X chromosome in a Chinese hamster cell background. The first fragmentation step involved the removal of the long arm together with ~750 kb of [alpha]-satellite DNA from the DXZ1 locus, leaving an array estimated to be ~2.5 Mb which stability assays revealed to have full mitotic function (22,23). In the second round of fragmentation, a hypoxanthine phosphoribosyltransferase (HPRT)-based telomere seeding construct was integrated randomly into proximal Xp. Molecular and cytogenetic analysis showed that this had resulted in the generation of a linear minichromosome <10 Mb in size that was as stable mitotically, in the Chinese hamster cell background, as the chromosome from which it was derived. The second breakpoint mapped between the gene ZXDA and DXS14. Based on physical and cytogenetic maps of the region, a straightforward breakage and healing event predicted the generation of a minichromosome of 3-4 Mb (24), while the structure recovered was at least twice this size. The disparity appears to be accounted for by amplification and rearrangement of the terminal DNA sequences, which may have arisen following breakage and prior to telomere healing (8,22).

In order to circumvent chromosomal rearrangements, which we have found frequently characterize truncated chromosomes rescued through randomly integrated telomere seeding constructs in this hybrid cell system, we have focused on the manipulation of human chromosomes using a targeted telomere-associated chromosome fragmentation approach. The feasibility of introducing a de novo telomere at a specific locus through homologous recombination was first demonstrated in the human HT1080 cell line (25). However, a major limitation is the low frequency of homologous recombination events in mammalian somatic cell lines. Recently, it has been demonstrated that efficient systematic manipulation of human chromosomes can be achieved following transfer of the chromosome of interest into the chicken B cell line DT40 (26). This vertebrate cell line is unusual in that it displays a highly recombination-proficient phenotype (27). In this report, we describe the targeted manipulation of a derivative human X chromosome present in a DT40-human microcell hybrid resulting in the generation of an ~2.4 Mb human X centromere-based minichromosome. This minichromosome has been characterized using both molecular and cytogenetic analyses, and its mitotic stability has been examined, both in DT40 and following transfer into hamster and human cell lines.

RESULTS

Generation of a DT40-human Xp microcell hybrid

A derivative human Xp chromosome present in the hamster-human hybrid HyTM1/36 and generated by a single round of chromosome fragmentation (22,23) was transferred into DT40 cells using microcell-mediated chromosome transfer (MMCT). One histidinolr/hygromycinr DT40 clone was recovered and designated IG4. Fluorescence in situ hybridization (FISH) revealed the presence in IG4 of a telocentric chromosome hybridizing with the human X [alpha]-satellite probe, DXZ1. No hamster chromosomes had been co-transferred. FISH mapping of human Xp-derived cosmids and the generation of an Alu-PCR paint revealed that material from Xp22 had been deleted during transfer (data not shown).

Targeting strategy for the generation of an X centromere-based human minichromosome

Our strategy was to remove the remaining human chromosome arm by targeting a de novo telomere into the gene ZXDA, which has been estimated cytogenetically to lie ~400 kb away from the [alpha]-satellite array (24). Based on the analysis of X chromosome breakpoints in patients with isochromosomes, the gene appears to be transcribed away from the centromere (H. Willard, personal communication). This information allowed us to design a single targeting/telomere seeding construct, which should fragment the chromosome at a defined point, while simultaneously stabilizing the fragmented centric product by seeding a new chromosome end. A cosmid clone containing ZXDA (ICRFc10000DO2100) has been identified previously (24). A restriction map was established and a 12.5 kb SstII-XhoI fragment encompassing the ZXDA open reading frame (ORF) was introduced into a plasmid carrying CMVneo, 2.3 kb of `Tag' DNA, plus 800 bp of (TTAGGG)n repeat. The targeting strategy is shown in Figure 1A.

   A
   B

Figure 1. (A) Strategy for generating a human X centromere-based minichromosome in DT40. (B) Southern blot analysis of drug-resistant clones. The DNA samples were digested with XhoI and KpnI and hybridized to the probes as shown. *Fragment representing the targeted allele.

Identification of homologous recombinants

The targeting/telomere seeding construct, pKZR1-4, was introduced into the DT40-human Xp hybrid, IG4, by electroporation. Two hundred G418r/hygromycinr clones were isolated and screened by Southern blotting. The restriction enzyme XhoI was used, together with the junction probe ZX5.5, to identify targeting events. Since IG4 is haploid for human ZXDA, a successful targeting event would predict the replacement of the ~21 kb XhoI fragment by an 18 kb hybridization signal. The Southern blot screen identified two potentially targeted clones, IKNEC12 and IKNFA3 (Fig. 1B). However, the screen was complicated by the detection of a doublet in the parental cell line and in all non-targeted transfectants. While in clone IKNFA3 both of these bands were absent, in IKNEC12 the upper band remained. The ZXDA locus is part of a much larger genomic duplication, with a second virtually identical locus, ZXDB, present ~500 kb telomeric on human Xp (24,28). The extent of the duplication has not been established. It appears that the junction probe used to detect recombination events detects a second related locus, presumably originating from the vicinity of the ZXDB gene. The seeding of a new telomere following a targeting event at the ZXDA locus would be predicted to result in loss of distal Xp sequences, as appears to have occurred in clone IKNFA3. However, even following a successful targeting and telomere seeding event, the displaced human Xp sequences may be retained within the cell as a result of translocation into the chicken genome. The persistence of the upper hybridization band in IKNEC12 suggests that this may have occurred in this particular cell line. The filters were stripped and rehybridized with: (i) ZX7: DNA included in the targeting construct, but specific for ZXDA. In IKNEC12 and IKNFA3, the `targeted' 18 kb band was detected, while in each of the non-targeted sibs both the ZXDA parental~21 kb band and a signal from the randomly integrated construct were present (15.5 kb where the intact construct has integrated). (ii) CMVneo: in most of the transfectants, including IKNEC12 and IKNFA3, a diffuse >7 kb XhoI terminal restriction fragment was detected, indicating that the construct has seeded a de novo telomere (Fig. 1B).

Cytogenetic analysis of the ZXDA targeted cell lines

The composition of the human chromosome in the targeted cell lines IKNFA3 and IKNEC12 was examined by FISH using the following probe DNAs: pHyTM, the original telomere seeding construct minus (TTAGGG)n; CMVNeo-Tag, a probe specific for pKZR1-4; and DXZ1, specific for human X [alpha]-satellite DNA. In both IKNFA3 and IKNEC12, all probes co-localized to a small, independently segregating dot chromosome (Fig. 2).


Figure 2. In situ hybridization of the minichromosome present in the IKNFA3. (A) A metaphase spread shows detection of CMVneo-Tag (which detects pKZR1-4) with FITC (green) and human X [alpha]-satellite DNA DXZ1 with Cy3 (red). (B) A metaphase spread hybridized with pHYTM[delta] (green) and DXZ1 (red). (C) Metaphase chromosomes probed with both CMV-Tag (green) and HyTM (red). The chromosomal DNA has been counterstained with To-pro-3-iodide (blue). Identical images were obtained for early passage IKNEC12 cells (data not shown).

Molecular analysis of experimentally seeded telomeres

To determine the status of both experimentally seeded telomeres, DNA from the targeted cell lines was examined by Bal31 nuclease digestions and Southern blotting. For cell line IKNFA3 (DNA extracted at generations 25, 60 and 125), terminal restriction fragments were detected with probe DNAs from both pHyTM1 and pKZR1-4 (Fig. 3 and data not shown). This suggests that the targeted human X chromosome in this cell line exists as a stable, linear molecule with telomere function provided by the two introduced (TTAGGG)n constructs. In IKNEC12, while a terminal XhoI restriction fragment was detected using the CMVneo probe on DNA extracted from cells at generation 20 (Fig. 1B), by generation 60 only discrete, Bal31-insensitive, restriction fragments could be detected (Fig. 3). Moreover, the DNA probe from pHyTM1 also hybridized to a discrete, Bal31-insensitive SstI fragment in DNA extracted from IKNEC12 at generation 60. These data indicate that the structure of the chromosome present in the targeted line IKNEC12 has changed during growth, such that the introduced (TTAGGG)n constructs no longer provide telomere function.


Figure 3. (A) A schematic representation of the modified X centromere-based chromosome showing XhoI and SstI terminal restriction fragments. (B) Bal31 nuclease digestions showing that pKZR1-4 and pHyTM1 are both providing telomere function in IKNFA3, while in IKNEC12 (generation 60) only discrete, Bal31-insensitive fragments are detected. DNA was digested with either XhoI (and probed with CMVneo from pKZR1-4) or SstI (and probed with HPH from pHyTM1).

Pulsed-field gel electrophoresis (PFGE) of the targeted chromosomes

To determine the overall size of the chromosomes present in IKNEC12 and IKNFA3, undigested high molecular weight (HMW) DNA from these and other cell lines was electrophoretically separated, transferred and probed with the human X [alpha]-satellite DNA, DXZ1. In Figure 4, no signal is detectable beyond the wells for the starting cell line, IG4, and the non-homologous recombinants IKNBA9 and IKNAG3. In cell line 36HB7-7, which carries an X-derived minichromosome of ~8 Mb (8), a hybridization signal, which has migrated just a few millimetres into the gel, can be detected. For IKNFA3, DNA preparations from different passage numbers consistently produced a hybridization signal just above the largest Saccharomyces cerevisiae chromosome (2.2 Mb). A similarly sized band was seen in DNA from IKNEC12 at generation 25. However, by generation 50 the ~2.4 Mb signal was noticeably weaker (Fig. 4), and was undetectable at later generations (data not shown). This indicates that targeting has produced a linear, human X centromere-based minichromosome of ~2.4 Mb in both cell lines, but only in IKNFA3 has this structure been stably maintained. We have been unable to resolve the chromosome present in later passage IKNEC12, and hence its size and structure (linear or ring) remain unclear.


Figure 4. PFGE of intact or NotI-digested DNA. HMW DNA was electrophoresed through a 0.7% agarose gel under the following conditions: 0.25× TBE at 11°C, 5000-1000 s pulse (logarithmically ramped), 110-100° linearly ramped electrode angle, at 50-45 V (ramped linearly) for 120 h. Markers were S.pombe, H.wingei and S.cerevisiae. The gel was transferred and probed with DXZ1 (human [alpha]-satellite DNA).

Molecular composition of the X-derived minichromosome present in IKNFA3

We previously have estimated, on the basis of restriction enzyme digests, that the size of the [alpha]-satellite array in cell line HyTM1/36 (which contains the Xp chromosome transferred to IG4, from which the minichromosome has been derived) to be ~2.5 Mb (23). A similar analysis of the [alpha]-satellite present in IKNFA3 found a total array length of ~1.8 Mb (Fig. 5A). To determine whether there had been any gross rearrangement of the [alpha]-satellite domain during generation of the minichromosome, a PFGE comparison was made (Fig. 5B). The two arrays appear to be identical and our present size estimate valid. Therefore, the bulk of the DNA present on the IKNFA3 minichromosome is [alpha]-satellite.


Figure 5. Molecular composition of the human minichromosome present in IKNFA3. (A) PFGE of the [alpha]-satellite array in IKNFA3. Genomic DNA was digested with various restriction enzymes and filters probed with DXZ1. Gel conditions were: 160 V, pulse parameters 120-200 s, in 0.5× TBE and 1% agarose for 45 h at 10°C. (B) Comparison of the [alpha]-satellite array in IKNFA3 with the parental chromosome present in hamster hybrid HyTM1/36. Gel conditions were as for (A) except that the gel was run at 14°C and 170 V. (C) Southern blot analysis of the [gamma]-satellite domain present in IKNFA3 compared with the parental chromosome in HyTM1/36. The filter was hybridized with the gamma-X DNA probe 2D12/E12. (D) PFGE of the proximal Xp region. DNA from IG4, IKNEC12 (generation 25) and IKNFA3 was digested with either NotI or EagI and the filter probed with (i) [gamma]-X and (ii) ZX1.7. Gel conditions were as for (A) above.

The only other satellite sequence assigned to the pericentromeric region of the human X chromosome is a GC-rich [gamma]-satellite repeat (29). Recently, we have shown that the [gamma]-satellite repeat is present as an ~40 kb block located on the short arm side of the centromere between ZXDA and DXZ1 (C. Lee, R. Critcher, W. Mills and C.J. Farr, manuscript in preparation). We therefore investigated whether, as would be predicted, the [gamma]-satellite domain is retained on the IKNFA3 minichromosome. For various restriction enzymes, an identical hybridization pattern was seen in IKNFA3 compared with HyTM1/36 and other human X-containing somatic cell hybrids (Fig. 5C and data not shown).

Many restriction enzymes which contain CpG dinucleotides within their recognition motif cut rarely, if at all, within the A+T-rich [alpha]-satellite domain (30). For example, both NotI and EagI release the [alpha]-satellite present at the human X centromere from which the minichromosome has been derived as a single megabase-sized restriction fragment (Fig. 4 and unpublished data); digestion of DNA from IG4, IKNFA3 and early-passage IKNEC12 with NotI revealed an [alpha]-satellite-hybridizing band similar in size to that seen with uncut HMW DNA from the minichromosome-carrying cell lines (Fig. 4). This indicates that any NotI sites present on the X centromere-based minichromosome must flank the [alpha]-satellite array. This ~2 Mb fragment also hybridized weakly with the [gamma]-satellite probe DNA (data not shown). Previous work has revealed that the [gamma]-satellite array has within it a single NotI-EagI site (C. Lee, R. Critcher, W. Mills and C.J. Farr, manuscript in preparation). Digestion of DNA from IKNFA3 and early-passage IKNEC12 with NotI alone also produced a [gamma]-satellite-hybridizing band of ~410 kb, while for IG4 a NotI fragment of ~1 Mb was identified (Fig. 5D). When the filter was stripped and reprobed with DNA from the ZXDA locus (ZX1.7), the same hybridization pattern was detected (Fig. 5D). This suggests that in the minichromosome-carrying lines, targeting a new chromosome end into the ZXDA locus has disrupted an ~1 Mb [gamma]-satellite-positive NotI restriction fragment, such that the remaining NotI site (which lies within the [gamma]-satellite array) now defines an~410 kb terminal restriction fragment. Various hybrid DNAs were also digested with EagI. An EagI site has been mapped immediately 5[prime] to the ZXDA ORF (proximal to the targeted locus). Probing with [gamma]-satellite DNA revealed an ~390 kb signal in all three hybrids (Fig. 5D). An EagI fragment of the same size hybridizes with the probe ZX1.7 (Fig. 5D).

From PFGE, we are unable to determine the precise distance between the [gamma]- and [alpha]-satellite arrays, but FISH on the intact parental X chromosome has indicated a distance of ~70 kb (C. Lee, R. Critcher, W. Mills and C.J. Farr, manuscript in preparation). The juxtaposition of the [gamma]- and [alpha]-satellite arrays on the IKNFA3 minichromosome was confirmed by FISH using mechanically stretched chromosomes and chromatin fibres (Fig. 6). The combined information obtained from these analyses suggests that the organization of known DNA sequences on the X centromere-based minichromosome present in IKNFA3 is as shown in Figure 7.


Figure 6. Juxtaposition of [gamma]- and [alpha]-satellite arrays on the X centromere-based minichromosome. (A and B) In situ hybridization of mechanically stretched chromosomes from IKNFA3 simultaneously probed with [alpha]- (green) and [gamma]-satellite (red). The chromosomal DNA is counterstained with To-pro-3-iodide (blue). (C) Chromosome fibres from IKNFA3 probed with [alpha]- and [gamma]-satellite DNA. No DNA counterstain has been applied.


Figure 7. Schematic representation of the organization of known DNA sequences on the human minichromosome present in IKNFA3.

Stability of the minichromosome in DT40 and after transfer to mammalian cell lines

The copy number of the minichromosome present in IKNFA3 was assessed by FISH at generation number ~25 using the [alpha]-satellite probe DXZ1. This revealed that 97% of IKNFA3 cells retained one copy of the minichromosome at this stage. The cell line was then split and the two sublines maintained independently for 100 generations. Aliquots of subline 1 were maintained either in G418 at high concentration (2 mg/ml) or without any selection, while aliquots of subline 2 were maintained in a lower G418 concentration (1 mg/ml) or again without selective agent. The stability of the minichromosome in the two sublines (both with and without selection) was assessed by FISH. For both IKNFA3 sublines maintained without selection, a majority of the metaphases retained either one or two copies of the minichromosome at all time points, with the percentage of minichromosome-containing metaphases in the two sublines declining by generation 100 to 92 and 88%, respectively (Table 1). The minichromosome loss rates per generation for the two sublines over the 100 generation time span were 0.63 and 0.07%, respectively. Very little variation in minichromosome copy number was observed, with most cells having either zero, one or two copies. Few integrations were detected. However, in the cells maintained with selection (in particular on the higher G418 concentration), there was an increase over time in the number of cells retaining two copies of the minichromosome.

Table 1. Mitotic stability of the IKNFA3 minichromosome
Cell line No. of generations without selection (total no. of generations) Total no. of metaphases examined (no. of 3n cells) Total no. of minichromos-omes (value per 100 cells) FISH analysis No. of cells with integrations Loss Rate (R)
No. of minichromosomes/cell
0 1 2 3 4 5 6 7 8 9 10 >10 (no. of m/c)
IKNFA3 0 (25) 100 (3) 103 (103) 0 97 3 0 0 0 0 0 0 0 0 0 0  
IKNFA3 0 (75) 100 (0) 135 (135) 1 63 36 0 0 0 0 0 0 0 0 0 0  
Subline 1 50 (75) 100 (0) 104 (104) 0 96 4 0 0 0 0 0 0 0 0 0 0  
IKNFA3 0 (125) 100 (4) 204 (204) 0 4 92 1 2 1 0 0 0 0 0 0 0 0.0063
Subline 1 100 (125) 100 (2) 108 (108) 8 78 13 0 1 0 0 0 0 0 0 0 0 (R* = -0.0005)
IKNFA3 0 (75) 100 (3) 108 (108) 3 88 10 0 0 0 0 0 0 0 0 0 0  
Subline 2 50 (75) 100 (2) 98 (98) 5 92 3 0 0 0 0 0 0 0 0 0 0  
IKNFA3 0 (125) 100 (2) 103 (103) 4 81 11 0 0 0 0 0 0 0 0 0 5 0.0007
Subline 2 100 (125) 100 (2) 96 (96) 12 80 8 0 0 0 0 0 0 0 0 0 0 (R* = 0.0007)
FA3HT3 0 (120) 76 (0) 386 (508) 0 0 8 11 10 18 11 9 4 4 1 0 6  
  90 (120) 104 (89) 228 (219) 12 28 22 18 13 5 3 1 0 0 0 0 10 0.0093
FA3HT3 0 (90) 106 (0) 501 (473) 0 0 7 13 35 19 21 4 5 1 1 0 3  
Subclone 7 60 (90) 114 (5) 388 (340) 12 13 19 18 23 16 7 6 2 0 0 1 (11) 8 0.0055
FA3HT 4-1 0 (120) 100 (2) 434 (434) 0 5 4 17 28 25 12 5 2 0 1 0 3  
  90 (120) 97 (0) 309 (319) 8 14 23 13 16 7 6 6 1 1 0 1 (16) 4 0.0034
FA3HT4-1 0 (90) 101 (6) 498 (493) 0 5 9 11 27 17 17 6 8 1 1 2 (11) 26  
Subclone 2 60 (90) 104 (7) 358 (344) 3 8 28 18 12 14 11 0 2 2 1 1 (12) 23 0.0060
FA3Wg 6-6 0 (50) 105 (101) 266 (253) 0 22 26 36 13 4 2 0 0 0 0 0 19  
  25 (50) 100 (100) 152 (152) 0 61 31 4 3 1 0 0 0 0 0 0 2 0.0202
FA3Wg 8-4 0 (50) 100 (80) 231 (231) 1 23 42 19 9 5 1 0 0 0 0 0 5  
  25 (50) 100 (32) 111 (111) 21 56 18 3 0 0 0 0 0 0 1 0 1 0.028
For the analysis of minichromosome loss rate (R), the number of minichromosomes in 100 metaphases was scored by FISH after the indicated number of generations growth with (n0) and without (e.g. n100) selection. R was calculated as follows: n100 = n0 × (1 - R)100. For the calculation of R*, the number of minichromosomes after 100 generations growth without selection was compared with the number at total generation 25 (the first time point analysed).
Minichromosome loss rates in other independently derived microcell hybrids not detailed above: HT1080 hybrids: FA3HT1-2, R = 0.0099; FA3HT2, R = 0.0040; FA3HT4-2, R = 0.0105; FA3HT5, R = -0.0005; Wg3H hybrids: FA3Wg3-2, R = 0.0079; FA3Wg4-3, R = 0.0148; FA3Wg7-6, R = 0.0092; FA3Wg8-2, R = 0.0307. In other FA3HT subclones: FA3HT3-5, R = 0.0022; FA3HT3-12, R = 0.0063; FA3HT4-1-13, R = 0.0118.

The IKNFA3 minichromosome was transferred, using MMCT, into both the Chinese hamster cell line Wg3H and the human cell line HT1080. Many hybrids were recovered and six of each type were expanded and analysed further. PFGE of uncut DNA from the human and hamster minichromosome hybrids showed that the X centromere-based minichromosome had been transferred as an intact ~2.4 Mb linear entity (Fig. 8). These hybrids, plus several subclones derived from them, were maintained with and without selection for several months. Data from FISH analyses are shown in Table 1 (data are presented in full for only a few hybrids; for most lines, only the minichromosome loss rate is presented). For both the hamster and human hybrids, the most striking observation was the variation in minichromosome copy number, ranging from one to eight (with occasional cells having >8 copies). The same variability in copy number was observed in hybrids that had been recloned. After prolonged growth without selection (25-90 generations, depending on the cell line), the minichromosome was maintained, although the copy number was again variable and occasional integration events were detected. It can be seen from Table 1 that the average copy number declines with growth without selection, with loss rates ranging from 0.22 to 3.07% in the different hybrids.


Figure 8. PFGE of intact HMW DNA isolated from various microcell hybrids. Uncut DNA was electrophoresed as described for Figure 4 and the gel transferred and probed with DXZ1.


DISCUSSION

Generation of a human minichromosome in DT40

There have now been several reports describing the generation of human minichromosomes. The approaches used fall into two broad categories: either manipulation of existing chromosomes, as described here, or the assembly of structures de novo following the introduction of large arrays of [alpha]-satellite DNA. This `de novo' approach has provided the strongest experimental support yet for [alpha]-satellite DNA playing a crucial role in mitotic centromere function. However, the products generated have often proved difficult to characterize fully, in terms of both their precise DNA content and structure. In general, these structures appear to consist of multimers of the ingoing DNA molecules adopting a final overall size in the 1-10 Mb range (4,5).

The generation of minichromosomes through the manipulation of existing human chromosomes has also been reported. In some cases, naturally occurring minichromosomes have been retrofitted with selectable markers (31), while others have experimentally fragmented non-essential chromosomes maintained in rodent-human somatic cell hybrids (8,9,11). A major limitation of the latter approach has been its reliance on fortuitous random integration of (TTAGGG)n constructs. Recently, it has been shown that it is possible to efficiently modify human chromosomes maintained in the recombinogenic chicken cell line DT40, opening up the possibility of systematically manipulating chromosomes in a way not feasible in mammalian somatic cell lines (26,32). Moreover, on the basis of the PCR-based telomeric repeat amplification protocol assay, DT40 cells are telomerase-positive (W. Mills and C.J. Farr, unpublished data), and (TTAGGG)n constructs seed de novo telomeres in DT40 very efficiently (33; unpublished data).

In this study, we describe the manipulation of a human Xp chromosome following transfer to DT40 and the generation of an ~2.4 Mb minichromosome. This chromosome is linear and has two artificially seeded telomeres: one immediately flanking the [alpha]-satellite array on what was the long arm side of the original X chromosome (the result of a random integration event prior to transfer to the DT40 cell) and one targeted to proximal Xp euchromatin, specifically the ZXDA locus. The frequency with which homologous recombinants were recovered (2/200) is on the low side for DT40, even when human chromosomal loci are targeted. However, the facts that (i) both simultaneous targeting and telomere seeding events are required; and (ii) the target locus, ZXDA, is part of a large genomic duplication, may both have contributed to a reduction in the observed targeting frequency.

Cytogenetic and molecular examination of the targeted cell lines at generation ~30 showed that a linear minichromosome was present in both. However, while this minichromosome has been stably maintained in IKNFA3, the structure of the modified chromosome in IKNEC12 changed during growth. FISH analysis revealed that a small independently segregating chromosome was present in IKNEC12 at late passage, but it appeared larger than the starting minichromosome, showed some size variability from cell to cell and could not be resolved by PFGE. While it is possible that the telomeres on the IKNEC12 minichromosome were deficient, leading to its instability, it is also conceivable that a rearrangement has arisen early during propagation of this transfectant which, due to a growth advantage, has allowed these cells to dominate the population. It should be possible to differentiate between these possibilities by recloning early passage IKNEC12 cells.

Molecular and cytogenetic examination of the linear FA3 minichromosome

The stable minichromosome present in IKNFA3 has been characterized extensively, at both the molecular and cytogenetic levels. Based on PFGE, this X centromere-based minichromosome is ~2.4 Mb in size, of which the bulk of the DNA is [alpha]-satellite. The [alpha]-satellite array at the human X centromere appears to consist of a single locus, DXZ1 (34-36). Extensive work by others has failed to find evidence for large (>100 kb) blocks of non-repetitive DNA, either interspersed with, or present as a large block in, the X [alpha]-satellite sequences (30,37). Between the DXZ1 locus and the gene ZXDA (at which point a new chromosome end has been created) lies a comparatively small (~40 kb) block of [gamma]-satellite repeat (29). We have not been able to identify any restriction enzyme which releases a fragment hybridizing to both repeats, other than the ~2 Mb NotI fragment and, therefore, by this means, we cannot estimate the distance between the [alpha]- and [gamma]-satellite arrays. However, by fibre FISH, the [gamma]-satellite DNA array does not appear to be immediately juxtaposed to the DXZ1 [alpha]-satellite array. Previous measurements on intact human X chromosomes have allowed us to estimate an ~70 kb gap between these two satellite DNAs (C. Lee, R. Critcher, W. Mills and C.J. Farr, manuscript in preparation). This may suggest that a different and undetermined X centromeric sequence lies between these two satellite arrays. Alternatively, the ~70 kb region may be comprised of highly diverged [alpha]- and/or [gamma]-satellite sequences. The distance between the [gamma]-satellite array and the ZXDA gene has been estimated, from EagI and NotI digests, as ~400 kb. This is consistent with the findings of Miller et al. (24) and Mahtani and Willard (30), who concluded that the edge of the [alpha]-satellite array must lie within a few hundred kilobases of unique Xp euchromatic sequence. The nature of the DNA sequences that lie between the ZXDA locus and the [gamma]-satellite array remains to be determined.

Mitotic stability of the FA3 minichromosome in DT40

Some 88-92% of IKNFA3 cells retain one or more copies of the human X centromere-based minichromosome after 100 generations without selection. Overall, a minichromosome loss rate per generation of 0.07-0.63% was estimated in the DT40 background when the minichromosome number in cells maintained without selection for 100 generations was compared with that in cells maintained with selection for the same period. If the number following growth without selection is compared instead with the number at generation 25 (the first time point analysed), the revised loss rates per division are 0.07% (unchanged) and -0.05% (reflecting a slight increase in the total number of minichromosomes). This difference reflects the tendency for IKNFA3 cells to accumulate more than one copy of the minichromosome when maintained with selection; this points to some degree of chromosome mis-segregation in DT40 cells, the apparent extent of which may be exaggerated as a result of growth advantage effects. Overall, it appears that the IKNFA3 minichromosome is very stable in the chicken cell background. Similar findings on mammalian minichromosome stability in DT40 have been reported by others (11).

Mitotic stability of the FA3 minichromosome in established human and rodent cell lines

The IKNFA3 minichromosome was microcell transferred into two established mammalian cell lines, Wg3H (an aneuploid Chinese hamster line) and HT1080 (a pseudodiploid human cell line). PFGE of several independent hybrids revealed that the~2.4 Mb minichromosome had been transferred as an intact linear structure. The most striking cytogenetic observation of both sets of hybrids was the variation in minichromosome copy number, ranging in general from one to eight copies within a single hybrid. This could arise from the transfer of multiple minichromosomes to a single recipient cell; however, the fact that so many independently derived hybrids display this phenotype makes it unlikely that this is the full explanation. To rule out the possibility that either the hybrids were not clonal or, alternatively, that some initial instability had existed following transfer from the chicken cell background, two of the FA3HT hybrids were recloned. When the subclones (several from each) were examined by FISH at generation ~25, the same variation in copy number was seen.

Several FA3Wg and FA3HT microcell hybrids were passaged with and without selection for prolonged periods of time. The bulk of cells in all hybrids retained at least one copy of the IKNFA3 minichromosome (for FA3Wg hybrids 79-100% and for FA3HT hybrids 88-100% after growth without selection for 25-90 generations, as indicated in Table 1). However, the total number of minichromosomes fell in virtually all hybrids, with loss rates ranging from 0.79 to 3.07% for the FA3Wg hybrids and from 0.22 to 1.05% (although one line showed a slight overall gain of 0.05%) in the FA3HT hybrids. These loss rates were generally higher than those observed in DT40, but are comparable with loss rates reported for various de novo minichromosomes generated in HT1080 [loss rates in the range of 0.48-1.6% (5,38) and 0.1-0.5% (4)].

Loss rates, copy number control and a lower size limit?

Although in all three cell backgrounds loss rates for the FA3 minichromosome are low, it does appear to be segregating more erratically in the mammalian cell background compared with DT40; this is most noticeable in terms of the copy number per cell. Earlier studies by us on (as far as we can tell from extensive molecular analyses) the same human X centromeric DNA, retained in a Chinese hamster cell line, revealed that when attached to the short arm of the human X chromosome (i.e. on a telocentric chromosome of ~50 Mb), it was retained at either one or two copies in 100% of cells after 60 days without selection. The same centromere when present on a truncated ~8 Mb minichromosome was retained in the hamster cell background at either one or two copies in 95% of cells when without selection for a similar period. The calculation of loss rates from previous data sets reveals that these chromosomes were maintained without any detectable loss (8,23). Although such assays are limited in their usefulness, the data suggest that the more recent manipulations of this Xp derivative have reduced slightly the overall stability of the modified chromosome in the mammalian cell background. This may reflect the existence of crucial cis-acting DNA sequences distal to ZXDA. However, the existence of naturally occurring Xq isochromosomes lacking ZXDA argues against this idea (unless compensatory DNA sequnces exist in proximal Xq) (39,40). Alternatively, the reduced stability could be linked to the fact that the overall size of the chromosome has been substantially reduced. Most naturally occurring human `dot' chromosomes, and the more recent de novo structures, have been estimated to be in the 5-10 Mb size range (4,5,41). This may reflect an overall greater stability of larger minichromosomes in mammalian cells (5,38). Moreover, the greater stability of this human minichromosome in DT40 might reflect the fact that the chicken genome has numerous small microchromosomes. However, some of the de novo chromosomes reported by Ikeno et al. (5) were estimated to be in the 1-2 Mb size range; these smaller minichromosomes, although exhibiting higher loss rates, were propagated in HT1080 in a copy number-controlled manner. At the moment, therefore, there is no clear explanation for the variable copy number of the FA3 minichromosome described here on transfer back into established mammalian cell lines. It is conceivable that this apparent semi-stability might reflect epigenetic effects following its passage through the chicken DT40 cell (42). Intriguingly, however, when the larger minichromosome present in IKNEC12 was transferred to HT1080, virtually all cells displayed zero, one or two copies, in marked contrast to the FA3HT hybrids (unpublished data). Although the structure of the chromosome in IKNEC12 has not been fully defined, it does not appear to have picked up any chicken DNA while in the DT40 background (on the basis of a total chicken genomic DNA paint) and is presumed to be the result of amplification/multimerization of the human DNA sequences, which would increase both the overall size and the amount of centromeric DNA. With the development of both more, well characterized, chromosome reagents and improved stability assays, questions concerning the requirements of an artificial mammalian chromosome (such as whether a lower size limit exists) can be addressed.

MATERIALS AND METHODS

Microcell-mediated chromosome transfer

The Xp derivative chromosome (present in cell line HyTM1/36) carries two dominant selectable markers: hygromycin B dehydrogenase (hygro) (associated with the experimentally seeded telomere and immediately juxtaposed with the [alpha]-satellite DNA) and histidinol dehydrogenase (hisD) (targeted to the MIC2 gene at Xp22.32), and is maintained in an HPRT-deficient Chinese hamster cell background (1,8,22,23). DT40 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) plus 10% fetal bovine serum (FBS) + 1% chicken serum + 4 mM L-glutamine (Gibco BRL, UK). To transfer the human chromosome into DT40 cells, the protocol of Dieken and Fournier was followed (43). Microcells were purified over Percoll gradients. Hybrids were selected for (and the recipient DT40 cells selected against) using 5 mM histidinol (Sigma, Poole, UK) and 500 µg (500 U)/ml hygromycin B (Calbiochem, UK). Any contaminating intact donor cells were eliminated using HAT selection (Gibco BRL). At least 2 × 107 recipient DT40 cells were used per fusion, with approximately three times as many microcells.

Transfer of the minichromosome from the DT40 cells to the Chinese hamster cell line Wg3H and the human cell line HT1080 was undertaken using standard MMCT technology (44). Wg3H and HT1080 derivatives carrying a puromycin resistance selectable marker were used as recipients (WgPAC and HTNEP), allowing puromycin selection to be used to kill any contaminating donor cells. Wg3H hybrids were selected for in the following medium: DMEM, 10% FBS, 7.5 µg/ml puromycin, 500 µg/ml hygromycin plus 2 mg/ml G418SO4. HT1080 hybrids were selected for in: DMEM, 10% FBS, 0.5 µg/ml puromycin, 300 µg/ml hygromycin plus 1 mg/ml G410SO4.

Recombinant DNA constructs

All molecular biology manipulations were carried out using standard methods (45). The ZXDA targeting/telomere seeding plasmid pKZR1-4 was constructed as follows: a 12.5 kb XhoI-SstII fragment from cZXDA (24), which spans the ZXDA gene, was cloned into XhoI + SstII-cut Bluescript (KSII-) (Stratagene, La Jolla, CA). An 800 bp array of (TTAGGG)n telomeric repeat (46), together with 2.3 kb of `Tag' DNA (genomic DNA originating from the vicinity of the human SOX4 locus) and a cytomegalovirus (CMV)-driven neor gene were cloned into pRS416 (Stratagene) such that they could be removed on a single 6.5 kb XhoI fragment, which was then introduced into the ZXDA-containing Bluescript vector described above. The final construct was linearized prior to transfection with NotI, which cuts immediately distal to the telomere repeat array.

Transfection of the human Xp-DT40 hybrid, IG4

The human Xp-DT40 hybrid IG4 was transfected with the targeting/telomere seeding construct pKZR1-4 by electroporation of 2 × 107 cells with 25 µg of NotI-linearized plasmid at 25 µF and 550 V using a Bio-Rad (Hercules, CA) Gene Pulser as described by Dieken and Fournier (43). After 24 h in basic growth medium, the cells were resuspended in selective medium [DT40 medium, 500 µg/ml hygromycin plus 2 mg/ml G418SO4 (Gibco BRL)] and aliquoted into four 96-well flat bottomed microtitre dishes (Falcon, Becton Dickinson, NJ). After 14 days, transfectant colonies were transferred to 12-well plates. Aliquots were harvested for small-scale DNA preparation and, following regrowth, 10% dimethyl sulfoxide and extra FBS were added and the plates frozen at -70°C. Following the Southern blot screen, clones of interest were thawed and expanded.

DNA preparation, digestion and analysis

DNA from cell lines and plasmids was prepared by standard methods (45). For the initial Southern blot screen of transfectants, small-scale DNA extractions on 3 ml of cell suspension were carried out (47). For the targeting screen, the diagnostic enzyme XhoI was used in conjunction with KpnI. KpnI does not cut within this region of the human genome and its use allowed the XhoI-digested DNAs to be resolved effectively through routine 0.5% agarose gels. Restriction digests were done according to the manufacturer's recommendations (Gibco BRL). Bal31 nuclease (New England BioLabs, MA) digestions were done as previously described (1). Southern transfer from 0.5% agarose gels was onto nylon membranes (Hybond N+; Amersham, Little Chalfont, UK). Filters were probed with DNA fragments labelled by the random primer method. Southern hybridizations were done in a Hybaid (UK) oven and washed at high stringency (0.1× SSPE/0.1% SDS) at 65°C unless otherwise stated.

Probes

Southern blot filters were probed with the following DNA fragments: ZX5.5, a 5.5 kb SstII-XhoI fragment from cZXDA (24) which spans the ZXDA CpG island and lies outside the block of homology present in the targeting construct; ZX7, a 6.5 kb EcoRI fragment from cZXDA which lies 3[prime] (and telomeric) to the ZXDA ORF and falls within the block of homology present in the targeting construct; ZX1.7, a 1.7 kb XhoI-BamHI fragment from cZXDA which lies ~23 kb 5[prime] (and centromeric) of the ZXDA ORF; pBR, a 2.1 kb EcoRI-NdeI from pBR322; CMVneo, a NotI-SmaI 2.3 kb fragment encompassing the CMV promoter and neo gene; DXZ1, an X chromosome-specific [alpha]-satellite 2 kb BamHI fragment from pSV2X5; HPH, a 550 bp EcoRI-SstII fragment from the hygromycin B resistance gene present in pSV2hygro (48); and 2D12/E2, a 1.2 kb EcoRI fragment from the [gamma]-satellite-containing cosmid clone CX16-2D12 (29).

In situ hybridization was done using the following probe DNAs: DXZ1, the human X chromosome [alpha]-satellite DNA (49); CX16-2D12, a human X [gamma]-satellite cosmid (29); HyTM[delta], a derivative of the telomere construct pHyTM1, which lacks the (TTAGGG)n array (1); and CMVneo-Tag, effectively pKZR1-4 which has been deleted for the (TTAGGG)n repeat, the plasmid backbone and the ZXDA homology. Additional cosmids used in characterization of IG4 were: cZXDA, a human X-derived cosmid containing the gene ZXDA (DXS422, ICRFc100G11100) (24); c5.1, a cosmid derived from the human MIC2 locus (50); and CY29, which maps immediately proximal to the telomere repeat at Xpter/Ypter (51). An Alu-PCR-derived paint was generated from IG4 DNA and used as a FISH probe onto normal human male metaphase spreads.

PFGE

Agarose blocks were prepared and digested according to standard methods. Digested DNA was electrophoresed through 1% ultrapure agarose (Bio-Rad) gels in 0.5× TBE using a Bio-Rad Chef-DRII apparatus. Uncut and NotI-cut DNA (Fig. 4) were separated using a Biometra Rotophor and 0.7% chromosomal grade agarose (Bio-Rad) in 0.25× TBE. PFGE run conditions were as indicated in the figure legends. Markers used were Schizosaccharomyces pombe chromosomes, Hansenula wingei chromosomes, Saccharomyces cerevisiae chromosomes and a lambda ladder (Bio-Rad and Biometra, Germany).

In situ hybridization

In situ hybridization to metaphase chromosomes and interphase nuclei from exponential cultures was as described previously (52). Images were visualized and digitally captured using a Nikon Optiphot microscope and a Bio-Rad MRC 600 confocal scanning laser microscope. Bio-Rad CoMOS version 7.0 system software was used to capture images, which were then merged in Adobe Photoshop 4.0.

Preparation of mechanically stretched chromosomes and chromosome fibres

Mechanically stretched chromosomes were prepared by the method of Haaf and Ward (53) with the following modifications. Actively dividing lymphoblastoid cells were incubated with colcemid for 3 h, washed in 1× phosphate-buffered saline A and incubated for 10 min in pre-warmed hypotonic (0.75 M) KCl at 37°C. The cells were placed on ice and their density adjusted to 1 × 104 cells/ml. A 500 µl aliquot was centrifuged onto ethanol-cleaned slides in a Cytospin 3 (Shandon, PA) at 800 r.p.m. for 4 min. The slides were fixed (3:1 methanol:acetic acid) for 15 min at room temperature and air dried. Slides were prepared on the day of use and baked for 2 h prior to hybridization.

Stretched chromosome fibres were prepared according to the method of Fuchs et al. (54), which is a modification of the method of Fidlerova et al. (55).

Mitotic stabilities

To assay mitotic stability, the DT40-derived lines were split 1:10 every other day to maintain exponential growth (the doubling time of these lines is ~12 h). Selective medium contained either 1 or 2 mg/ml G418SO4 (as specified in the text). The HT1080 (FA3HT) and Wg3H (FA3Wg) lines were passaged 1:10 every third day (average doubling time is ~24 h). For long-term growth with selection, the FA3HT hybrids were passaged in the presence of 300 µg/ml hygromycin and 500 µg/ml G418SO4, while the FA3Wg hybrids were grown in 500 µg/ml hygromycin and 1 mg/ml G418SO4. Minichromosome loss rates were calculated as described by Ikeno et al. (5).

ACKNOWLEDGEMENTS

We thank Tony Jackson (Cambridge) for kindly providing us with the DT40 cell line, Tom Ebersole (Edinburgh) for providing the puromycin-resistant HT1080 cell line HTNEP, Jean-Marie Buerstedde (Basel) for selection cassettes for use in DT40 cells, Keith Fournier and Ellen Dieken (Seattle) for very valuable advice on microcell-mediated chromosome transfer into DT40 cells, and Hunt Willard (Cleveland) for supplying unpublished information concerning the human ZXDA locus. This work was supported by the Medical Research Council (MRC) of Great Britain. C.L. was a postdoctoral fellow of the Natural Sciences and Engineering Council (NSERC) of Canada; C.J.F. is an MRC Senior Research Fellow.

REFERENCES

1. Farr, C., Fantes, J., Goodfellow, P. and Cooke, H. (1991) Functional reintroduction of human telomeres into mammalian cells. Proc. Natl Acad. Sci. USA, 88, 7006-7010. MEDLINE Abstract

2. Barnett, M.A., Buckle, V.J., Evans, E.P., Porter, A.C.G., Rout, D., Smith, A.G. and Brown, W.R.A. (1993) Telomere directed fragmentation of mammalian chromosomes. Nucleic Acids Res., 21, 27-36. MEDLINE Abstract

3. Hanish, J.P., Yanowitz, J.L. and de Lange, T. (1994) Stringent sequence requirements for the formation of human telomeres. Proc. Natl Acad. Sci. USA, 91, 8861-8865. MEDLINE Abstract

4. Harrington, J.J., Van Bokkelen, G., Mays, R.W., Gustashaw, K. and Willard, H.F. (1997) Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nature Genet., 15, 345-355. MEDLINE Abstract

5. Ikeno, M., Grimes, B., Okazaki, T., Nakano, M., Saitoh, K., Hoshino, H., McGill, N.I., Cooke, H. and Masumoto, H. (1998) Construction of YAC-based mammalian artificial chromosomes. Nature Biotechnol., 16, 431-439.

6. Brown, K.E., Barnett, M.A., Burgtorf, C., Shaw, P., Buckle, V. and Brown, W.R.A. (1994) Dissecting the centromere of the human Y chromosome with cloned telomeric DNA. Hum. Mol. Genet., 3, 1227-1237. MEDLINE Abstract

7. Aladjem, M.I., Groudine, M., Brody, L.L., Dieken, E.S., Fournier, R.E., Wahl, G.M. and Epner, E.M. (1995) Participation of the human beta-globin locus control region in initiation of DNA replication. Science, 270, 815-819. MEDLINE Abstract

8. Farr, C.J., Bayne, R.A.L., Kipling, D., Mills, W., Critcher, R. and Cooke, H.J. (1995) Generation of a human X-derived minichromosome using telomere-associated chromosome fragmentation. EMBO J., 14, 5444-5454. MEDLINE Abstract

9. Heller, R., Brown, K.E., Burgtorf, C. and Brown, W.R.A. (1996) Mini-chromosomes derived from the human Y chromosome by telomere directed chromosome breakage. Proc. Natl Acad. Sci. USA, 93, 7125-7130. MEDLINE Abstract

10. Willard, H.F. (1996) Chromosome manipulation: a systematic approach toward understanding human chromosome structure and function. Proc. Natl Acad. Sci. USA, 93, 6847-6850. MEDLINE Abstract

11. Shen, M.H., Yang, Y., Loupart, M.-L., Smith, A. and Brown, W. (1997) Human mini-chromosomes in mouse embryonal stem cells. Hum. Mol. Genet., 6, 1375-1382. MEDLINE Abstract

12. Huxley, C. (1994) Mammalian artificial chromosomes: a new tool for gene therapy. Gene Ther., 1, 7-12. MEDLINE Abstract

13. Brown, W.R.A., Heller, R., Loupart, M.-L., Shen, M.-H. and Chand, A. (1996) Mammalian artificial chromosomes. Curr. Opin. Genet. Dev., 6, 281-288.

14. Wilkie, A.O.M., Lamb, J., Harris, P.C., Finney, R.D. and Higgs, D.R. (1990) A truncated human chromosome 16 associated with alpha thalassaemia is stabilized by the addition of telomeric tepeat (TTAGGG)n. Nature, 346, 868-871. MEDLINE Abstract

15. Lamb, J., Harris, P.C., Wilkie, A.O.M., Wood, W.G., Dauwerse, J.H.G. and Higgs, D.R. (1993) De novo truncation of chromosome 16p and healing with (TTAGGG)n in the alpha-thalassemia mental-retardation syndrome (ATR-16). Am. J. Hum. Genet., 52, 668-676. MEDLINE Abstract

16. Flint, J., Wilkie, A.O.M., Buckle, V.J., Winter, R.M., Holland, A.J. and McDermid, H.E. (1995) The detection of subtelomeric chromosomal rearrangements in idiopathic mental retardation. Nature Genet., 9, 132-140. MEDLINE Abstract

17. duSart, D., Cancilla, M.R., Earle, E., Mao, J., Saffrey, R., Tainton, K.R., Kalitsis, P.M., Martyn, J., Barry, A.E. and Choo, K.H.A. (1997) A functional neocentromere formed through activation of a latent human centromere and consisting of non-alpha satellite DNA. Nature Genet., 16, 144-153. MEDLINE Abstract

18. Choo, K.H.A. (1997) Centromere DNA dynamics: latent centromeres and neocentromere formation. Am. J. Hum. Genet., 61, 1225-1233. MEDLINE Abstract

19. Dobbs, D.L., Shaiu, W.-L. and Benbow, R.M. (1994) Modular sequence elements associated with origin regions in eukaryotic chromosomal DNA. Nucleic Acids Res., 22, 2479-2489. MEDLINE Abstract

20. Boulikas, T. (1996) Common structural features of replication origins in all life forms. J. Cell. Biochem., 60, 297-316. MEDLINE Abstract

21. Willard, H.F. (1998) Centromeres: the missing link in the development of human artificial chromosomes. Curr. Opin. Genet. Dev., 8, 219-225. MEDLINE Abstract

22. Farr, C.J., Stevanovic, M., Thomson, E.J., Goodfellow, P.N. and Cooke, H.J. (1992) Telomere-associated chromosome fragmentation: applications in genome manipulation and analysis. Nature Genet., 2, 275-282. MEDLINE Abstract

23. Bayne, R.A.L., Broccoli, D., Taggart, M.H., Thomson, E.J., Farr, C.J. and Cooke, H.J. (1994) Sandwiching of a gene within 12 kb of a functional telomere and alpha satellite does not result in silencing. Hum. Mol. Genet., 3, 539-546. MEDLINE Abstract

24. Miller, A.P., Gustashaw, K., Wolff, D.J., Rider, S.H., Monaco, A.P., Eble, B., Schlessinger, D., Gorski, J.L., van Ommen, G.J.B., Weissenbach, J. and Willard, H.F. (1995) Three genes that escape X chromosome inactivation are clustered within a 6 Mb YAC contig and STS map in Xp11.21-p11.22. Hum. Mol. Genet., 4, 731-739. MEDLINE Abstract

25. Itzhaki, J.E., Barnett, M.A., MacCarthy, A.B., Buckle, V.J., Brown, W.R.A. and Porter, A.C.G. (1992) Targeted breakage of a human chromosome mediated by cloned human telomeric DNA. Nature Genet., 2, 283-287. MEDLINE Abstract

26. Dieken, E.S., Epner, E.M., Fiering, S., Fournier, R.E.K. and Groudine, M. (1996) Efficient modification of human chromosomal alleles using recombination-proficient chicken/human microcell hybrids. Nature Genet., 12, 174-182. MEDLINE Abstract

27. Buerstedde, J.-M. and Takeda, S. (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell, 67, 179-188. MEDLINE Abstract

28. Greig, G.M., Sharp, C.B., Carrel, L. and Willard, H.F. (1993) Duplicated zinc finger protein genes on the proximal short arm of the human X chromosome: isolation, characterisation and X-inactivation studies. Hum. Mol. Genet., 2, 1611-1618. MEDLINE Abstract

29. Lee, C., Li, X., Jabs, E.W., Court, D.R. and Lin, C.C. (1995) Human gamma X satellite DNA: an X chromosome specific centromeric DNA sequence. Chromosoma, 104, 103-112. MEDLINE Abstract

30. Mahtani, M.M. and Willard, H.F. (1998) Physical and genetic mapping of the human X chromosome centromere: repression of recombination. Genome Res., 8, 100-110. MEDLINE Abstract

31. Raimondi, E., Balzaretti, M., Moralli, D., Vagnarelli, P., Tredici, F., Bensi, M. and De Carli, L. (1996) Gene targeting to the centromeric DNA of a human minichromosome. Hum. Gene Ther., 7, 1103-1109. MEDLINE Abstract

32. Koi, M., Lamb, P.W., Filatov, L., Feinberg, A.P. and Barrett, J.C. (1997) Construction of chicken-human microcell hybrids for human gene targeting. Cytogenet. Cell Genet., 76, 72-76. MEDLINE Abstract

33. Kuroiwa, Y., Shinohara, T., Notsu, T., Tomizuka, K., Yoshida, H., Takeda, S.-i., Oshimura, M. and Ishida, I. (1998) Efficient modification of a human chromosome by telomere-directed truncation in high homologous recombination-proficent chicken DT40 cells. Nucleic Acids Res., 26, 3447-3448. MEDLINE Abstract

34. Yang, T.P., Hansen, S.K., Oishi, K.K., Ryder, O.A. and Hamkalo, B.A. (1982) Characterisation of a cloned repetitive DNA sequence concentrated on the human X chromosome. Proc. Natl Acad. Sci. USA, 79, 6593-6597. MEDLINE Abstract

35. Willard, H.F., Smith, K.D. and Sutherland, J. (1983) Isolation and characterization of a major tandem repeat family from the human X chromosome. Nucleic Acids Res., 11, 2017-2033.

36. Waye, J.S. and Willard, H.F. (1985) Chromosome-specific alpha satellite DNA: nucleotide sequence analysis of the 2.0 kilobasepair repeat from the human X chromosome. Nucleic Acids Res., 13, 2731-2743.

37. Mahtani, M.M. and Willard, H.F. (1990) Pulsed-field gel analysis of [alpha]-satellite DNA at the human X chromosome centromere: high frequency polymorphisms and array size estimate. Genomics, 7, 607-613. MEDLINE Abstract

38. Masumoto, H., Ikeno, M., Nakano, M., Okazaki, T., Grimes, B., Cooke, H. and Suzuki, N. (1999) Assay of centromere function using a human artificial chromosome. Chromosoma, 107, 406-416.

39. Wolff, D.J., Miller, A.P., van Dyke, D.L., Schwatz, S. and Willard, H.F. (1996) Molecular definition of breakpoints associated with human Xq isochromosomes: implications for mechanisms of formation. Am. J. Hum. Genet., 58, 154-160. MEDLINE Abstract

40. James, R.S., Dalton, P., Gustashaw, K., Wolff, D.J., Willard, H.F., Mitchell, C. and Jacobs, P.A. (1997) Molecular characterization of isochromosome of Xq. Ann. Hum. Genet., 61, 485-490. MEDLINE Abstract

41. Carine, K., Jacquemin-Sablon, A., Waltzer, E., Mascarello, J. and Scheffler, I.E. (1989) Molecular characterization of human minichromosomes with centromere from chromosome 1 in human-hamster hybrid cells. Somat. Cell. Mol. Genet., 15, 445-460. MEDLINE Abstract

42. Karpen, G.H. and Allshire, R.C. (1997) The case for epigenetic effects on centromere identity and function. Trends Genet., 13, 489-496. MEDLINE Abstract

43. Dieken, E.S. and Fournier, R.E.K. (1996) Homologous modification of human chromosomal genes in chicken B-cell × human microcell hybrids. In Fournier, R.E.K. (ed.), Methods. Academic Press, Vol. 9, pp. 56-63. MEDLINE Abstract

44. Schafer, A.J. and Farr, C.J. (1998) Somatic cell hybrid approaches to genome mapping. In Spurr, N. (ed.), ICRF Handbook of Genome Analysis. Blackwell Science, Chapter 14, pp. 321-365.

45. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

46. de Lange, T., Shiue, L., Myers, R.M., Cox, D.R., Naylor, S.L., Killery, A.M. and Varmus, H.E. (1990) Structure and variability of human chromosome ends. Mol. Cell. Biol., 12, 4834-4843.

47. Laird, P.W., Zijderveld, A., Linders, K., Rudnicki, M.A., Jaenisch, R. and Berns, A. (1991) Simplified mammalian DNA isolation procedure. Nucleic Acids Res., 19, 4293. MEDLINE Abstract

48. Kaster, K.R., Burgett, S.G., Rao, R.N. and Ingolia, T.D. (1983) Analysis of a bacterial hygromycin B resistance gene by transcriptional and translational fusions and by DNA sequencing. Nucleic Acids Res., 11, 6895-6911. MEDLINE Abstract

49. Wolfe, J., Darling, S.M., Erickson, R.P., Craig, I.W., Buckle, V.J., Rigby, P.W.J., Willard, H.F. and Goodfellow, P.N. (1985) Isolation and characterisation of an alphoid centromeric repeat family from the human Y chromosome. J. Mol. Biol., 182, 477-485. MEDLINE Abstract

50. Smith, M.J., Goodfellow, P.J. and Goodfellow, P.N. (1993) The genomic organisation of the human pseudoautosomal gene MIC2 and the detection of a related locus. Hum. Mol. Genet., 2, 417-422. MEDLINE Abstract

51. Cooke, H.J., Brown, W.R.A. and Rappold, G.A. (1985) Hypervariable telomeric sequences from the human sex chromosomes are pseudoautosomal. Nature, 317, 687-692. MEDLINE Abstract

52. Palin, A.H., Critcher, R., Fitzgerald, D.J., Anderson, J.N. and Farr, C.J. (1998) Direct cloning and analysis of DNA sequences from a region of the Chinese hamster genome associated with aphidicolin-sensitive fragility. J. Cell Sci., 111, 1623-1634. MEDLINE Abstract

53. Haaf, T. and Ward, D.C. (1994) Structural analysis of [alpha]-satellite DNA and centromere proteins using extended chromatin and chromosomes. Hum. Mol. Genet., 3, 697-709. MEDLINE Abstract

54. Fuchs, C., Liehr, T. and Rautenstrauss, B. (1997) High resolution FISH of stretched chromosome fibers. Trends Journal Technical Tips Online (http://www.biomednet.com/db/tto ).

55. Fidlerova, H., Senger, G., Kost, M., Sanseau, P. and Sheer, D. (1994) Two simple procedures for releasing chromatin from routinely fixed cells for fluoresence in situ hybridization. Cytogenet. Cell Genet., 65, 203-205. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +44 1223 333972; Fax: +44 1223 333992; Email: c_farr@mole.bio.cam.ac.uk

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