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Regional Mapping Strategies Utilizing Microcell Hybrids
METHODS: A Companion to Methods in Enzymology 9, 20–29 (1996) Article No. 0004
Regional Mapping Strategies Utilizing Microcell Hybrids Teresa L. Johnson-Pais* and Robin J. Leach*,† The University of Texas Health Science Center at San Antonio, Departments of *Cellular and Structural Biology and †Pediatrics, 7703 Floyd Curl Drive, San Antonio, Texas 78284
Microcell hybrids are useful resources for the mapping of human chromosomes. The procedure of microcell-mediated chromosome transfer often causes fragmentation of the donor chromosome. These fragment-containing microcell hybrids frequently contain a limited region around the locus used in selecting for retention of the chromosome in the hybrids, as well as other fragments from the donor chromosome. Monochromosomal microcell hybrids are useful as the donor cell line for creation of radiation-reduced hybrids. In contrast to fragmentcontaining microcell hybrids, radiation-reduced hybrids can be used to construct maps of regions of chromosomes that lack selectable markers. For both fragment-containing hybrids and radiation-reduced hybrids, the presence or absence of chromosome-specific sequences can be determined and used to construct a linear map of the chromosome. A protocol and general overview outlining the key concepts in the construction and analysis of a radiation-reduced hybrid panel is presented. q 1996 Academic Press, Inc.
The mapping of genes and arbitrary DNA sequences to particular chromosomes or chromosomal regions in the human genome has been greatly facilitated by the use of somatic cell genetic techniques. Somatic cell hybrids particularly useful for chromosomal localization have been constructed by the method of microcell-mediated chromosome transfer (MMCT) (1, 2) as described in Killary and Lott (2A). MMCT generally transfers single or a few intact chromosomes from one cell line to another; however, rearrangement of the donor chromosomes can occur, resulting in fragment-containing microcell hybrids (3, 4). It has been suggested that the number of fragment-containing microcell hybrids obtained in an experiment is related to both the conditions used for donor cell micronucleation and the cells chosen as the recipient fusion partner (3, 4). Several published articles have demonstrated the feasibility of mapping human chromosomes using fragment-containing microcell hybrids. Leach et al. (5) de-
scribed physical mapping of human chromosome 17 using a panel of hybrids derived by MMCT of human chromosome 17 into rat hepatoma recipient cells. Thirty-six rat 1 human microcell hybrids were analyzed with a series of chromosome 17-specific markers. Seven clones appeared to retain an intact chromosome 17, while 29 hybrids retained subchromosomal fragments. In several hybrids, these fragments were noncontiguous. A linear order for the markers that minimized the number of breaks in the hybrids was derived and was consistent with the known gene map. Jeggo et al. (6) transferred human chromosome 2 from a mouse 1 human monochromosomal hybrid line into a recipient hamster line, which resulted in the isolation of a panel of hybrids bearing chromosome 2 fragments of various sizes, due to the frequent breakage of chromosome 2 upon transfer into hamster cells. From these hybrids, an order was derived for a series of human chromosome 2q markers. Darmoul et al. (7) were also able to utilize fragment-containing microcell hybrids to order genes on human chromosome 2q21– q31 and to regionally localize the human DPP4 gene, which encodes dipeptidyl peptidase IV. The single requirement for successful MMCT is that the chromosome to be transferred contains a selectable marker. The marker can be either an endogenous biochemical marker [such as the nucleotide salvage pathway enzymes hypoxanthine phosphoribosyl transferase (HPRT), adenine phosphoribosyl transferase, or thymidine kinase], or an exogenously introduced, dominant selectable marker. The presence of a marker enables the chromosome to be selectively retained in the microcell hybrid. If a donor cell line contains only a single integration event of the selectable marker, most of the fragment-containing hybrids will contain a limited chromosomal region around the selectable marker’s integration site. It is important to note that since these fragments still contain a selectable marker, it is feasible to transfer these fragments to additional cell lines. Chromosome-mediated gene transfer (CMGT) is an-
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1046-2023/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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other method of introducing chromosomal fragments for regional mapping studies (8–10). In this transfection method, coprecipitates between calcium phosphate and isolated mitotic chromosomes are applied to recipient cells, and hybrids are selected by the presence of a selectable marker on the donor chromosome. This method appears to be useful as a tool for regional mapping, since cotransfer of syntenic loci occurs. However, frequent multiple interstitial events appear to contribute to chromosomal rearrangement in the introduced fragments. Porteus (9) was unable to perform any CMGT experiment that maintained the normal physical linkage of markers. He attributed this observation to deletion and religation events that occurred during the transfection process. Additionally, Pritchard and Goodfellow (10) tested the integrity of human X chromosome fragments transferred by CMGT and again observed frequent interstitial deletions. This type of chromosomal rearrangement precludes CMGT from providing adequate resources for mapping specific portions of the human genome. In contrast to fragment-containing microcell hybrids and CMGT hybrids, radiation-reduced hybrids provide a means for isolating human chromosomal fragments that lack selectable markers. These hybrids are constructed by irradiation of monochromosomal hybrid lines with subsequent fusion to a rodent ‘‘rescue’’ recipient cell line. Radiation-reduced hybrids are the only type of somatic cell hybrids that allow for high-resolution mapping of an entire chromosome. Placement of genes and markers using these types of hybrids requires extensive statistical analysis. The resolution of the maps depends on the fragmentation of chromosomes by X rays or g rays and is correlated with the physical distance (reviewed in 11–13). The basis for creating a linear order of markers along a chromosome relies on the theory that markers located close together should be rarely disrupted when the chromosome is fragmented by irradiation, while more distant markers will be more frequently disrupted. Radiation-reduced hybrid mapping, along with other somatic cell hybrid mapping methods, allows for placement of both polymorphic and monomorphic markers, unlike genetic mapping methods. Radiation-reduced hybrid maps constructed from irradiation of monochromosomal hybrid donor cells have been created for most of the human chromosomes and have provided excellent cloning resources for isolating sequences around the loci of interest (Table 1). Since this method does not rely on direct selection of the human chromosomal component in the hybrid, there is a relatively equal representation of all regions of the chromosome in the radiation-reduced hybrids; thus, maps that span the entire chromosome can be created. This chapter briefly reviews the methods used in constructing radiation-
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reduced hybrids from microcell hybrid donors for the purpose of creating high-resolution chromosome maps.
RADIATION-REDUCED HYBRIDS: BACKGROUND Goss and Harris (50, 51) performed early experiments with radiation-reduced hybrids that laid the groundwork for gene mapping with these types of hybrids. Their experiments utilized human diploid cells that were irradiated at dosages between 1000 and 4000 rads and subsequently fused to a hamster cell line deficient in the enzyme HPRT. Radiation-reduced hybrids were selected for activity of the human HPRT gene in medium containing hypoxanthine-aminopterin-thymidine (52); therefore, all the hybrids contained the portion of the human X chromosome encoding HPRT. After analyzing the hybrids for the presence of three additional X-linked genes, it was demonstrated that increasing doses of radiation greatly diminished cotransfer of other X-linked genes with the HPRT locus. These authors proposed that these genes became separated from the HPRT locus after radiation-induced chromosome fragmentation. Without selection to retain other portions of the X chromosome, these fragments would be lost as the cell population expanded. This observation was described as radiation-induced segregation of syntenic genetic loci. From these data, Goss and Harris (50, 51) determined an order for these four genes that was consistent with orders deduced from X chromosome translocations. In addition, a means to measure the relative physical distance between the markers was devised. If two loci are separated by a large distance, then it is more probable that radiation will be able to fragment the DNA between the loci and separate them. The number of events that cause segregation of one locus from another is also directly proportional to the radiation dosage. For many years the radiation-reduced hybrid methods of Goss and Harris were largely unused, partly due to the low density of markers available. By the mid 1980s, a renewed interest in these methods had developed. An important rebirth of radiation-reduced hybrid mapping appeared in 1990 (11). In this paper, a radiation-reduced hybrid panel for human chromosome 21 was constructed and analyzed with 14 chromosome 21specific markers. The creation of a map for chromosome 21 was achieved by the development of readily available computer programs that determined a map order based on the likelihood of one order versus alternative orders. An estimate of the distances between the markers was also deduced with these programs. The radiation-reduced hybrid map of chromosome 21 generated by Cox et al. (11) was compared to physical maps de-
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A9(neo3/t)-5 X A9 GM7297 X14-150 Q314-2 X UrdA 9TK X GM459 HHW661 X UCW113 HD113.2B X CHTG49 HHW416 X UCW113 HHW661 X UCW113 HHW661 X UCW113 I-7 X RJK459 R21/B1 X A23 or W3GH 706-B6 clone 17 X ATS-49tg GM10156B X ATS-49tg 640-63a12 X W3GH PK87-9 X CHTG49 640-63a12 X CHTG49 762-8A X W3GH J1-11 X CHOK1 J1 X 380-6 J1 X 380-6 J1 X 380-6 J2 X 380-6 M28 X Wg3-h RJ83.1FT X RJKM PCTBA1.89 X W3gH or RAG 7AE-4 X GM459 7AE-4 X GM459 X11-4A-1d-F-e X Don/a3 20XP3542-1-4 X UV20 and 20XP3542-1-4 X Wg3h 153-E9A X adeC 153-E7BX X adeC CHG3 X GM459 CHG3 X GM459 EYEF3A6 X 380-6 C12D X W3GH GM0616 X A23 GM0616 X A23 GM7297 X B14-150
3 3(X)b 3 4 4(5)b 4 4 5(4)b 5(4)b 6 6 8 8 9 9 9 10 11 11 11 11 11 12 16 17 17 17 18 19 Hu – CH X CH Hu – CH X CH Hu – CH X CH Hu – CH X CH Hu – Ha X Ha Hu – CH X CH Hu – CH X CH Hu – CH X CH Hu – Ha X CH
Hu – M X M Hu – Ha X CH Hu – CH X C Hu – CH X CH Hu – CH X CH Hu – M X CH Hu – CH X CH Hu – CH X CH Hu – CH X CH Hu – Ha X CH Hu – Ha X CH Hu – Ha X Ha Hu – CH X Ha Hu – Ha X CH Hu – Ha X CH Hu – Ha X CH Hu – CH X CH Hu – Ha X CH Hu – Ha X Ha Hu – Ha X Ha Hu – Ha X Ha Hu – Ha X Ha Hu – M X CH Hu – Ha X Ha Hu – M X CH or M Hu – R X CH Hu – R X CH Hu – CH X CH Hu – CH X Ha
Species
3 8 – 10 8 8 8 6, 20, 50 8 8 2.5 – 25
2–8 2.2 – 25 6 6 6.5 0.5 – 8 6.5 6.5 6.5 7 40 10 5 40 1–8 4–8 50 4–8 9 9 9 9 40 7 10 8 3–6 7 5
Dose (krads)
g g X X X X g g g
X g X X g g g g g g X g g X g X X g X X X X X g X g g g g
g or X
10/18c 5 103 103 85/130c 29 67 67 86
96 86 18/72c 72/160c 109/226c 12 101/134c 109/226c 109/266c 65/93c 4/40c 11/47c 97 23 53/250c 6/47c 28 12 102 102 100 86/102 17/60c 233 38/100c 76 44/61c 98/108c 19/83c
No. RH
7 7 14 28 18 5 14 32 22
25 7 3 7 11 10 15 13 18 15 21 47 6 19 17 21 9 56 16 30 32 299 10 38 22 22 35 91 27
31 – 96a 4 – 29 11 – 22 30 – 60 22 – 28 8 – 92a 21 – 27 16 – 28 15 – 21 Up to 60 Up to 100a 9 – 82 11 – 16 4 – 48 9 – 100a 0 – 100a 4 – 54 100a 21 – 39 18 – 29 14 – 24 14 – 57 12 – 59 8 – 83 8 – 45 44 – 67 31 – 60 6 – 65 NAa NAa NAa 32 – 59 32 – 59 17 – 42 NA 22 – 70 22 – 70 4 – 29
No. loci
Retention (%)
Note. Hu, human; CH, Chinese Hamster; Ha, Hamster; M, mouse; R, rat; Hc, human chromosome; RH, radiation hybrid; NA, not available. a Hybrids selected for human DNA. b A(B), donor line carried A:B translocation, markers scored on chromosome A. c Number of hybrids analyzed/number of hybrids isolated.
21 21 21 21 22 X X X X(3)b
Parental lines
HC
Human Chromosome-Specific Radiation-Reduced Hybrid Panels
TABLE 1
44 44 11 45 46 47 48 49 15
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31, 32 33 34 35 36 37 38 39 40 41 42 43
Reference
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rived from pulsed-field gel electrophoresis. They demonstrated that the physical distances estimated by radiation hybrid mapping were proportional to the physical distances obtained from pulsed-field gels. Other intensive statistical programs are now available to enable investigators to construct linear maps from their radiation-reduced hybrid data. One of the most widely used programs is RHMAP (53), which is discussed later in the text. Since the first few mapping experiments that utilized radiation-reduced hybrids, the majority of maps created with these hybrids have been constructed by irradiating monochromosomal microcell hybrids containing a single human chromosome in a rodent cell background. These irradiated cells are subsequently fused to another rodent cell line that is deficient in an endogenous selectable gene. The major advantage of radiation-reduced hybrids constructed from monochromosomal microcell hybrids, compared to whole genome radiation hybrids constructed from diploid cells, is that they contain human chromosomal fragments derived from a single chromosome. Because of the reduced complexity, the hybrids provide a useful source of enriched human DNA for cloning experiments. Another advantage of radiation-reduced hybrids is their lack of either retention bias around a selectable marker (excluding those radiation-reduced hybrids isolated by selecting for the presence of a selectable marker) or extensive chromosomal deletion and rearrangement like that observed with CMGT hybrids (9, 10).
CONSTRUCTION OF RADIATION-REDUCED HYBRIDS Construction of radiation-reduced hybrids requires lethal irradiation of donor cells with either X rays or g rays, which induces double-strand breaks in the chromosomes. Irradiation of the cells is performed on ice so that DNA repair is minimized (50). The irradiated cells are subsequently fused to recipient cells using polyethylene glycol or inactivated Sendai virus. The recipient cells generally have a deficiency in an endogenous selectable gene that allows hybrids to be isolated in a selective medium, following the introduction of the chromosome fragment containing a normal copy of the selectable gene. When rodent 1 human monochromosomal microcell hybrids are used as the donor cell line, the normal copy of the selectable gene is usually encoded in the rodent genome. This allows for random retention of all regions of the human chromosome without preferential retention of any human locus. If a dominant selectable marker has been introduced into the human chromosome, hybrids can be isolated by selecting on this marker. However, this causes biased reten-
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tion of this region in the hybrid panel and makes it difficult to construct a map in this region (19). Choice of Parental Cell Lines The first consideration in preparing to construct radiation-reduced hybrids is the choice of cell lines for fusion partners. Most of the radiation-reduced hybrid panels appearing in the literature have been produced with hamster cell recipients (see Table 1). In addition, the majority of chromosome-specific hybrids utilized as donor cell lines contain human chromosomes in a hamster background. Irradiated human 1 hamster microcell hybrids fused to a hamster recipient are generally believed to be the best combination of fusion partners, since hamster cell lines can exist in an aneuploid state with up to four chromosome complements (32). Our laboratory has had considerable success in preparing radiation-reduced hybrid panels for human chromosomes 3 (54) and 17 (41) by fusing irradiated monochromosomal hybrids in a rat hepatoma background to the hamster cell line GM459 (NIGMS Human Genetic Mutant Cell Repository, Camden, NJ), a cell line deficient in HPRT. It is evident from other reports that not all combinations of donor and recipient cell lines produce hybrids (11, 40). It is difficult to predict whether two cell lines will be optimal fusion partners; therefore, the fusogenic properties of cells must be determined empirically. Radiation Dose The next consideration in constructing a radiationreduced hybrid panel involves the choice of dose and source of the radiation used to fragment the chromosomes. Using monochromosomal microcell hybrids as donor cell lines, it is our experience that both the length and the copy number of the chromosome in the hybrid should be considered when selecting the radiation dose. Our experience also suggests that there is much more extensive chromosomal fragmentation when equivalent doses of radiation are given from a g source compared to an X-ray source. This may be due, in part, to the difficulty of delivering accurate doses with many X irradiators. Although higher doses of radiation increase map resolution, low-retention frequencies may occur, resulting in no significant linkage between markers. Retention frequencies are calculated by counting the number of hybrids scored as positive for a particular marker divided by the total number of hybrids in the panel. Theoretically, the maximum amount of information can be obtained in a panel exhibiting a retention frequency of 50% (55, 56). The dose of radiation applied to the donor cells can be chosen to increase or decrease the number of breaks along the chromosome and between markers by using data obtained from similar radiation hybrid experiments. It has been reported that by increasing the g radiation dose from
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5000 to 25,000 rads, marker retention frequencies drop from 27 to 3%, and retained fragment sizes decrease 5- to 10-fold (15). Size of the Hybrid Panel It is also important to ascertain the number of hybrids necessary to construct a map. If the retention frequency of markers in the panel is 20–45%, then a panel of 100 hybrids is necessary to construct a linear map order (57). Sample size guidelines proposed by Lunetta and Boehnke (56) require 100 hybrids to order 16 markers with a retention frequency of 20–50% with high probability, assuming that the markers are relatively evenly spaced. If the markers are not equidistant from each other, 100 hybrids are still sufficient to order markers with 1000:1 odds, again assuming that the retention frequency is in the range of 50%. Analysis of only 50 hybrids results in lower probabilities that the predicted order is the true order. In our experience, it is best to isolate at least 130 hybrid clones from a radiation-reduced hybrid fusion. These extra clones will enable the investigator to discard hybrids that have no human DNA, those that contain an entire chromosome, or those that are duplicate clones. In this regard, it is good experimental technique to isolate only one to three hybrid clones from any one flask to eliminate the possibility of obtaining identical clones.
10. Aspirate supernatant and add 0.5 ml 50% polyethylene glycol 1500 (Boehringer Mannheim, Indianapolis, IN) prewarmed to 377C. 11. After 1 min, add serum-free medium dropwise at the rate of 1 ml for the first minute and 2 ml per minute thereafter for a total of 2 min for a final volume of 5 ml. 12. Centrifuge fused cell suspension at 1000 rpm for 5 min. Aspirate supernatant and gently resuspend fused pellet in 5 ml of prewarmed (377C) serum-free medium. 13. Incubate fused cell suspension at 377C for 90 min with occasional mixing. 14. Centrifuge cell suspension at 1000 rpm for 5 min, aspirate supernatant, and resuspend pellet in 10 ml of appropriate complete medium containing appropriate selection. 15. Plate fused cells at 1 1 106 cells per 75-cm2 flask containing complete medium plus selective reagent. Plate irradiated donor control cells in three 75-cm2 flasks at 1 1 106 cells/flask each in complete medium. Plate nonirradiated recipient cells in three 75-cm2 flasks at 1 1 106 cells/flask each in complete medium plus selective agent. 16. Replace medium in flasks 72 h after fusion and then every 5–7 days for approximately 3 weeks, after which time hybrids are clonally isolated and expanded for high-molecular-weight DNA isolation.
GENERAL PROTOCOL FOR CONSTRUCTING RADIATION-REDUCED HYBRIDS
RADIATION-REDUCED HYBRID ANALYSES
1. Expand donor and recipient cell lines to approximately 1.5 1 107 cells each. 2. Harvest donor cells and obtain cell count. 3. Aliquot 1.5 1 107 donor cells into a conical centrifuge tube. Centrifuge cells at 1000 rpm for 5 min. 4. Aspirate supernatant and resuspend cell pellet in 10 ml of serum-free medium (Dulbecco’s modified Eagle’s medium or other basic cell growth medium). 5. Place 10 ml of donor cells to be irradiated into a sterile 25-cm2 flask. If cells are to be irradiated with a g source, the cells must be placed on ice due to the length of exposure time. Irradiate the donor cells at the determined dose. 6. If using g irradiation, the recipient cells can be harvested and counted while the donor cells are being irradiated. Retain 1.5 1 107 recipient cells. 7. After irradiation of the donor cells, remove aliquots containing 1.5 1 106 cells from both the irradiated donor cells and the nonirradiated recipient cells and save for control flasks. 8. Mix 1 1 107 cells of both donor and recipient together in a sterile 15-ml conical centrifuge tube. 9. Centrifuge mixed cells at 1000 rpm for 5 min.
In most radiation-reduced hybrids, multiple human DNA fragments are integrated into rodent chromosomes, but free fragments may also be observed (12). These fragments may or may not be contiguous. Phillipe et al. (58) analyzed the rearrangement of chromosomes in radiation-reduced hybrids constructed by irradiating a human 1 hamster monochromosomal hybrid fused to a mouse recipient cell line. They detected the rejoining of human chromosomal fragments with hamster chromosomal fragments and rarely detected human fragments integrated into the recipient mouse chromosomes. They were also able to isolate small human–hamster fragments that were retained as minichromosomes. Any apparently ‘‘intact’’ hamster chromosomes probably resulted from random rearrangement of irradiated fragments. Although relatively even retention frequencies are observed for most markers along a chromosome, many radiation-reduced hybrid panels exhibit unequal retention of markers located near the centromere (reviewed in 13). Both centromeric and telomeric fragments must play an essential role in the formation of these new ‘‘chromosomes’’ from broken chromosomal fragments.
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Fluorescence in situ hybridization (FISH) analyses have proven useful for detecting the number of human fragments retained in each clone. Total human DNA is biotin-labeled by nick-translation and hybridized to metaphase chromosome spreads from each clone (59). The human chromosomal fragments are subsequently detected by an avidin–fluorescein isothiocyanate conjugate, and rodent chromosomes are counterstained with propidium iodide (Fig. 1A). Side´n et al. (15) have observed the retention of one to eight human chromosomal fragments, regardless of the applied radiation dose. Francke et al. (42) analyzed numerous radiationreduced hybrids by FISH and demonstrated that 9 of 17 hybrids exhibited clonal heterogeneity, with retention of one to five fragments of human DNA. InterAlu polymerase chain reactions (PCR) (60) can also be performed with DNA isolated from each clone, with the resulting products biotin-labeled by nick-translation (61). These PCR products can then be hybridized to normal human metaphase chromosome spreads and detected with an avidin–FITC conjugate (Fig. 1B). This type of FISH analysis, sometimes referred to as reverse chromosome painting, will provide a cytogenetic evaluation of both the number of human fragments in the clone and the chromosomal regions from which the fragments were derived. Since the human chromosomal fragments in radiation-reduced hybrids are not selectively retained and can be lost after passage in culture (12, 25), it is very important that a large quantity of DNA be isolated prior to the analysis of marker content. We have found that regrowing the radiation-reduced hybrids to obtain additional DNA results in a significant number of hybrids with altered scoring for PCR markers. These alterations can be a result of both the loss of particular sequences and the apparent acquisition of new sequences, presumably related to subpopulations of cells with differential rates of growth. Since radiation-reduced hybrids can serve as a source of region-specific sequences, segregation of the unstable fragments can be exploited in an attempt to separate the fragment of interest from the other contaminating fragments in the hybrid. By single-cell subcloning, new cell populations that contain a less complex human component can be isolated. Radiation-reduced hybrids have been used to map a variety of markers. Early studies analyzed the presence or absence of DNA markers by Southern blotting techniques (11, 14, 15, 17, 23, 30, 37, 38, 43, 45–48). More recently, PCR methods have been utilized to analyze most markers. It has been well documented that PCR is a much more sensitive detection assay than Southern analysis. Since radiation-reduced hybrids are heterogeneous populations with multiple unstable fragments, which may be lost from the clone during expansion of the cells, combining the results from anal-
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yses by Southern techniques and PCR may not be equivalent. PCR may detect a sequence that is not present in the majority of cells in the population. For example, DNA from a radiation-reduced hybrid panel for human chromosome 18, analyzed by both Southern blotting and PCR methods, demonstrated a 4% discrepancy rate in scoring between the two methods (42). The major discrepancy in scoring was observed in hybrids that were negative by Southern analysis and positive by PCR analysis. PCR also has the advantage of requiring much less DNA for analysis, which is of great benefit when confronting the expansion of a hybrid clone for large-scale DNA isolation. We perform radiation-reduced hybrid PCR analyses in 96-well microtiter plates in an Omnigene thermocycler (Hybaid Limited, Middlesex, England). Each reaction is repeated in duplicate, and the clones are scored as positive, negative, or uncertain. Any hybrid scoring that does not correlate between the two plates should be retyped, although analysis packages tolerate incomplete data. Before any markers are scored through the hybrids, the human content of each hybrid can be analyzed by inter-Alu PCR amplification (60). We have performed this type of amplification and have found hybrid clones that do not appear to contain human DNA by this type of analysis; however, when individual markers are scored, these hybrids have indeed retained human fragments. With most of our panels, we have chosen to proceed with marker analysis without scoring the hybrids for human DNA content. Only after preliminary analysis with a subset of markers are hybrids discarded based on lack of human DNA, retention of the entire chromosome, or presence of duplicated clones. After careful checking, data are entered into a spreadsheet format for analysis using the appropriate statistical packages.
STATISTICAL ANALYSES The first analysis of marker scoring should be a twopoint pair-by-pair test of each marker against all the other markers. This is used to determine linkage groups, estimate locus retention probabilities, and estimate distances between markers. These data are then analyzed by several available methods relying on multipoint statistical approaches (56). To better demonstrate the radiation-reduced hybrid mapping approach, we will briefly describe our experience with mapping the gene for glucose-dependent insulinotropic polypeptide on human chromosome 17 (62). The TWOPOINT package of Cox et al. (11) was used to generate two-point data that provided lod (logarithm of the likelihood ratio for linkage) scores, u values (frequency of breakage), and distance (D) between two
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FIG. 1. (A) Total human DNA was biotin-labeled and hybridized to a metaphase chromosome spread of a radiation-reduced hybrid constructed by fusing a human 1 rat hybrid containing human chromosome 17 with a hamster cell line. Three human fragments were observed in this hybrid. (B) Inter-Alu PCR was performed on DNA isolated from a human 1 rat 1 hamster radiation-reduced hybrid containing fragments of human chromosome 17. The PCR products were biotin-labeled and hybridized to a normal diploid human metaphase spread. One chromosome 17 fragment was observed in this hybrid.
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markers, expressed in centirays. A distance of 1 cR6000 equals 1% of breakage between markers after exposure to 6000 rads of g radiation. With these data, pairs of markers with significant linkage were identified, and a preliminary map order was determined based on minimizing the sum of distances between markers. The next step entailed measuring the relative likelihood of one map order versus alternate orders in which the two internal markers are inverted (Fig. 2). The FOURPOINT package (11) calculates the likelihood of 12 possible orders from a set of four markers and lists the orders from most to least likely. This program provides rapid output, but is not ideal for long-range estimates of map order with more than four markers at a time, since the odds for ordering are based on internal inversions. The radiation-reduced hybrid mapping programs by Boehnke et al. (RHMAP) (53) are excellent statistical packages that combine two-point analysis with both minimum break and maximum likelihood analyses. The minimum break method uses only straightforward computations and is rapidly performed, although there are no estimates of the physical distances between markers, and it does not allow comparison of probabilities of one order versus an alternative order. Maximum likelihood provides estimates of physical distances between markers, but assumes that fragments are retained independently. These calculations are more complex and require longer computer running times. This method does allow the comparison of orders according to relative maximum likelihood. The RHMAP program can determine a map order for 40 or more markers at a time. The map is built on clusters of markers that exhibit linkage at 1000:1 odds; however, the support for order within these clusters is often not as strong. The last step in building a robust radiation-reduced hybrid map should consist of reevaluation of the marker data under the best map order based on an equal retention or centromeric retention model. Since many radiation-reduced hybrid panels demonstrate increased centromeric retention, computer simulations have been performed to compare the probability of cor-
FIG. 2. A radiation-reduced hybrid map of five loci on human chromosome 17. Distances are shown in cR6000 . Odds for order are given against the inversion of adjacent markers.
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rectly ordering markers assuming equal marker retention versus a centromeric retention model (56). The probabilities that the true order was the best order, based on equal retention or centromeric retention, were almost equal. Reevaluation of marker data should consist of examining hybrids with incomplete typing for a number of markers or a large number of breaks under the best map order. These hybrids should either be eliminated from the statistical analyses or retyped for all the markers and the data entry rechecked.
SUMMARY Methods for localizing sequences to small regions of the genome are important for increasing the resolution of physical maps of chromosomes. Radiation-reduced hybrids have provided an excellent means for creating high-resolution physical maps of markers along a chromosome. This method creates fragments that demonstrate little evidence for interstitial deletions or complex rearrangements, thus maintaining the synteny of loci along the chromosome. Radiation-reduced hybrid panels are not available for all the human chromosomes; however, human whole genome radiation-reduced hybrid mapping panels, created from diploid human fibroblast donors, have been constructed to regionally map markers from every human chromosome (63). DNA isolated from a human whole genome panel is currently available from Research Genetics (Huntsville, AL). This technology is also applicable to mapping genes and arbitrary DNA sequences in other species. In fact, radiation-reduced hybrid mapping panels have been created by investigators localizing sequences in the mouse (reviewed in 13). One disadvantage to the fragments that are created by this method is that they generally do not contain selectable markers and are not transferable to other cell lines by MMCT. However, a novel method has been introduced that allows the isolation of subchromosomal transferable fragments from any human chromosome utilizing MMCT and radiation-reduced fragmentation. In this strategy, Koi et al. (64) transfect a mammalian selectable marker into rodent cells that contain a single independently selectable human chromosome. This marked human chromosome is transferred by MMCT, which is followed by selection for both the human chromosome and the marker gene. Hybrid clones that survive the double selection will have a human chromosome tagged with both selectable markers. Microcells are prepared from these hybrids, which are subsequently irradiated and then fused to recipient cells. Hybrids that contain diverse fragments of the original human chromosome will be generated based on the various integration sites of the transfected selectable marker.
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Radiation-reduced hybrid mapping provides an integrated map for all types of markers, including arbitrary sequence-tagged sites, genetic markers, transcribed sequences, and centromeric and telomeric sequences. Markers are informative in every hybrid that is typed, and the loci do not need to be polymorphic. The amount of radiation that is used to fragment the donor chromosome(s) is a variable that can be controlled and that can be tailored to the specific experimental purposes. Radiation-reduced hybrids enable the construction of maps that bridge the gap between genetic maps and yeast artificial chromosome contig maps. With as few as 100 hybrids, data can be generated to create highresolution maps of an entire chromosome.
ACKNOWLEDGMENTS The authors thank Dr. Michael Siciliano for Fig. 1B, and Tracey B. Lewis for the data from Fig. 2. We also thank Dr. David Cox and Dr. Michael Boehnke for their radiation-reduced hybrid mapping statistical packages and Dr. Charles Leach for critical review of the manuscript.
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15. Side´n, T. S., Kumlien, J., Schwartz, C. E., and Ro¨hme, D. (1992) Somat. Cell Mol. Genet. 18, 33–44. 16. Atchison, L., Comis, R. L., and Atchison, M. L. (1994) Gene 151, 321–324. 17. Cox, D. R., Pritchard, C. A., Uglum, E., Casher, D., Kobori, J., and Myers, R. M. (1989) Genomics 4, 397–407. 18. Altherr, M. R., Plummer, S., Bates, G., MacDonald, M., Taylor, S., Lehrach, H., Frischauf, A.-M., Gusella, J. R., Boehnke, M., and Wasmuth, J. J. (1992) Genomics 13, 1040–1046. 19. Doucette-Stamm, L. A., Riba, L., Handelin, B., Difilippantonio, M., Ward, D. C., Wasmuth, J. J., Gusella, J. F., and Housman, D. E. (1991) Somat. Cell Mol. Genet. 17, 471–480. 20. Winokur, S. T., Schutte, B., Weiffenbach, B., Washington, S. S., McElligott, D., Chakravarti, A., Wasmuth, J. J., and Altherr, M. R. (1993) Am. J. Hum. Genet. 53, 874–880. 21. Warrington, J. A., Hall, L. V., Hinton, L. M., Miller, J. N., Wasmuth, J. J., and Lovett, M. (1991) Genomics 11, 701–708. 22. Warrington, J., Bailey, S. K., Armstrong, E., Aprelikova, O., Alitalo, K., Dolganov, G. M., Wilcox, A. S., Sikela, J. M., Wolfe, S. F., Lovett, M., and Wasmuth, J. J. (1992) Genomics 13, 803– 808. 23. Zoghbi, H. Y., McCall, A. E., and LeBorgne-Demarquoy, F. (1991) Genomics 9, 713–720. 24. Ragoussis, J., Jones, T. A., Sheer, D., Shrimpton, A. E., Goodfellow, P. N., Trowsdale, J., and Ziegler, A. (1991) Genomics 10, 598–607. 25. Sapru, M., Gu, J., Gu, X., Smith, D., Yu, C.-E., Wells, D., and Wagner, M. (1994) Genomics 21, 208–216. 26. Bookstein, R., Levy, A., MacGrogan, D., Lewis, T. B., Weissenbach, J., O’Connell, P., and Leach, R. J. (1994) Genomics 24, 317–323. 27. Florian, F., Hornigold, N., Griffin, D. K., Delhanty, J. D., Sefton, L., Abbott, C., Jones, C., Goodfellow, P. N., and Wolfe, J. (1991) Somat. Cell Mol. Genet. 17, 445–453. 28. Jackson, C. L., Britt, D. E., Graw, S. L., Potts, A., Santoro, K., Buckler, A. J., Housman, D. E., and Mark, H. F. L. (1992) Somat. Cell Mol. Genet. 18, 285–301. 29. Henske, E. P., Ozelius, L., Anderson, M. A., and Kwiatkowski, D. J. (1992) Genomics 13, 841–844. 30. Goodfellow, P. J., Povey, S., Nevanlinna, H. A., and Goodfellow, P. N. (1990) Somat. Cell Mol. Genet. 16, 163–171. 31. Rose, E. A., Glaser, T., Jones, C., Smith, C. L., Lewis, W. H., Call, K. M., Minden, M., Champagne, E., Bonetta, L., Yeger, H., and Housman, D. E. (1990) Cell 60, 495–508. 32. Glaser, T., Rose, E., Morse, H., Housman, D., and Jones, C. (1990) Genomics 6, 48–64. 33. Richard, C. W., III, Withers, D. A., Meeker, T. C., Maurer, S., Evans, G. A., Myers, R. M., and Cox, D. R. (1991) Am. J. Hum. Genet. 49, 1189–1196. 34. Richard, C. W., III, Boehnke, M., Berg, D. J., Lichy, J. H., Meeker, T. C., Hauser, E., Myers, R. M., and Cox, D. R. (1993) Am. J. Hum. Genet. 52, 915–921. 35. Richard, C. W., III, Cox, D. R., Kapp, L., Marnane, J., Cornelis, F., Julier, C., Lathrop, G. M., and James, M. R. (1993) Genomics 17, 1–5. 36. James, M. R., Richard, C. W., III, Schott, J.-J., Yousry, C., Clark, K., Bell, J., Terwilliger, J. D., Hazan, J., Dubay, C., Vignal, A., Agrapart, M., Imai, T., Nakamura, Y., Polymeropoulos, M., Weissenbach, J., Cox, D. R., and Lathrop, G. M. (1994) Nature Genet. 8, 70–76. 37. Sinke, R. J., Suijkerbuijk, R. F., Herbergs, J., Janssen, H., Cassiman, J. J., and Geurts van Kessel, A. (1992) Genomics 12, 206– 213.
AP: Methods
MAPPING STRATEGIES WITH MICROCELL HYBRIDS 38. Ceccherini, I., Romeo, G., Lawrence, S., Breuning, M. H., Harris, P. C., Himmelbauer, H., Frischauf, A. M., Sutherland, G. R., Germino, G. G., Reeders, S. T., and Morton, N. E. (1992) Proc. Natl. Acad. Sci. USA 89, 104–108. 39. Black, D. M., Nicolai, H., Borrow, J., and Solomon, E. (1993) Am. J. Hum. Genet. 52, 702–710. 40. Abel, K. J., Boehnke, M., Prahalad, M., Ho, P., Flejter, W. L., Watkins, M., VanderStoep, J., Chandrasekharappa, S. C., Collins, F. S., Glover, T. W., and Weber, B. L. (1993) Genomics 17, 632–641. 41. O’Connell, P., Albertsen, H., Matsunami, N., Taylor, T., Hundley, J. E., Johnson-Pais, T. L., Reus, B., Lawrence, E., Ballard, L., White, R., and Leach, R. J. (1994) Am. J. Hum. Genet. 54, 526–534. 42. Francke, U., Chang, E., Comeau, K., Geigl, E.-M., Giacalone, J., Li, X., Luna, J., Moon, A., Welch, S., and Wilgenbus, P. (1994) Cytogenet. Cell Genet. 66, 196–213. 43. Brook, J. D., Zemelman, B. V., Hadingham, K., Siciliano, M. J., Crow, S., Harley, H. G., Rundle, S. A., Buxton, J., Johnson, K., Almond, J. W., Housman, D. E., and Shaw, D. J. (1992) Genomics 13, 243–250. 44. Graw, S., Davidson, J., Gusella, J., Watkins, P., Tanzi, R., Neve, R., and Patterson, D. (1988) Somat. Cell Mol. Genet. 14, 233– 242. 45. Burmeister, M., Kim, S., Price, E. R., de Lange, T., Tantravahi, U., Myers, R. M., and Cox, D. R. (1991) Genomics 9, 19–30. 46. Frazer, K. A., Boehnke, M., Budarf, M. L., Wolff, R. K., Emanuel, B. S., Myers, R. M., and Cox, D. R. (1992) Genomics 14, 574– 584. 47. Benham, R., Hart, K., Crolla, J., Bobrow, M., Francavilla, M., and Goodfellow, P. N. (1989) Genomics 4, 509–517. 48. Gorski, J. L., Boehnke, M., Reyner, E. L., and Burright, E. M. (1992) Genomics 14, 657–665.
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49. Gorski, J. L., Burright, E. M., Reyner, E. L., Goodfellow, P. N., and Burgess, D. L. (1992) Genomics 14, 649–656. 50. Goss, S. J., and Harris, H. (1975) Nature 255, 680–684. 51. Goss, S. J., and Harris, H. (1977) J. Cell Sci. 25, 17–37. 52. Littlefield, J. W. (1964) Science 145, 709–710. 53. Boehnke, M., Lange, K., and Cox, D. R. (1991) Am. J. Hum. Genet. 49, 1174–1188. 54. Leach, R. J., Neal, M., Taylor, T., Reus, B., Hayes, S., Linn, R., O’Connell, P., and Naylor, S. L. (1995) Cytogent. Cell Genet. 68, 140–141. 55. Lange, K., and Boehnke, M. (1992) Ann. Hum. Genet. 56, 119– 144. 56. Lunetta, K., and Boehnke, M. (1994) Genomics 21, 92–103. 57. Barrett, J. H. (1992) Genomics 13, 95–103. 58. Phillipe, C., Nguyen, V. C., Slim, R., Holvoet-Vermaut, L., HorsCayla, M. C., and Bernheim, A. (1993) Hum. Genet. 92, 11–17. 59. Pinkel, D., Straume, T., and Gray, J. W. (1986) Proc. Natl. Acad. Sci. USA 83, 2934–2938. 60. Nelson, D. L., Ledbetter, S. A., Corbo, L., Victoria, M. F., RamıB rez-Solis, R., Webster, T. D., Ledbetter, D. H., and Caskey, C. T. (1989) Proc. Natl. Acad. Sci. USA 86, 6686–6690. 61. Liu, P., Siciliano, J., Seong, D., Craig, J., Zhao, Y., deJong, P. J., and Siciliano, M. J. (1993) Cancer Genet. Cytogenet. 65, 93–99. 62. Lewis, T. B., Saenz, M., O’Connell, P., and Leach, R. J. (1994) Genomics 19, 589–591. 63. Walter, M. A., Spillett, D. J., Thomas, P., Weissenbach, J., and Goodfellow, P. (1994) Nature Genet. 7, 22–28. 64. Koi, M., Johnson, L. A., Kalikin, L. M., Little, P. F. R., Nakamura, Y., and Feinberg, A. P. (1993) Science 260, 361–364.
AP: Methods
Regional Mapping Strategies Utilizing Microcell Hybrids Teresa L. Johnson-Pais* and Robin J. Leach*,† The University of Texas Health Science Center at San Antonio, Departments of *Cellular and Structural Biology and †Pediatrics, 7703 Floyd Curl Drive, San Antonio, Texas 78284
Microcell hybrids are useful resources for the mapping of human chromosomes. The procedure of microcell-mediated chromosome transfer often causes fragmentation of the donor chromosome. These fragment-containing microcell hybrids frequently contain a limited region around the locus used in selecting for retention of the chromosome in the hybrids, as well as other fragments from the donor chromosome. Monochromosomal microcell hybrids are useful as the donor cell line for creation of radiation-reduced hybrids. In contrast to fragmentcontaining microcell hybrids, radiation-reduced hybrids can be used to construct maps of regions of chromosomes that lack selectable markers. For both fragment-containing hybrids and radiation-reduced hybrids, the presence or absence of chromosome-specific sequences can be determined and used to construct a linear map of the chromosome. A protocol and general overview outlining the key concepts in the construction and analysis of a radiation-reduced hybrid panel is presented. q 1996 Academic Press, Inc.
The mapping of genes and arbitrary DNA sequences to particular chromosomes or chromosomal regions in the human genome has been greatly facilitated by the use of somatic cell genetic techniques. Somatic cell hybrids particularly useful for chromosomal localization have been constructed by the method of microcell-mediated chromosome transfer (MMCT) (1, 2) as described in Killary and Lott (2A). MMCT generally transfers single or a few intact chromosomes from one cell line to another; however, rearrangement of the donor chromosomes can occur, resulting in fragment-containing microcell hybrids (3, 4). It has been suggested that the number of fragment-containing microcell hybrids obtained in an experiment is related to both the conditions used for donor cell micronucleation and the cells chosen as the recipient fusion partner (3, 4). Several published articles have demonstrated the feasibility of mapping human chromosomes using fragment-containing microcell hybrids. Leach et al. (5) de-
scribed physical mapping of human chromosome 17 using a panel of hybrids derived by MMCT of human chromosome 17 into rat hepatoma recipient cells. Thirty-six rat 1 human microcell hybrids were analyzed with a series of chromosome 17-specific markers. Seven clones appeared to retain an intact chromosome 17, while 29 hybrids retained subchromosomal fragments. In several hybrids, these fragments were noncontiguous. A linear order for the markers that minimized the number of breaks in the hybrids was derived and was consistent with the known gene map. Jeggo et al. (6) transferred human chromosome 2 from a mouse 1 human monochromosomal hybrid line into a recipient hamster line, which resulted in the isolation of a panel of hybrids bearing chromosome 2 fragments of various sizes, due to the frequent breakage of chromosome 2 upon transfer into hamster cells. From these hybrids, an order was derived for a series of human chromosome 2q markers. Darmoul et al. (7) were also able to utilize fragment-containing microcell hybrids to order genes on human chromosome 2q21– q31 and to regionally localize the human DPP4 gene, which encodes dipeptidyl peptidase IV. The single requirement for successful MMCT is that the chromosome to be transferred contains a selectable marker. The marker can be either an endogenous biochemical marker [such as the nucleotide salvage pathway enzymes hypoxanthine phosphoribosyl transferase (HPRT), adenine phosphoribosyl transferase, or thymidine kinase], or an exogenously introduced, dominant selectable marker. The presence of a marker enables the chromosome to be selectively retained in the microcell hybrid. If a donor cell line contains only a single integration event of the selectable marker, most of the fragment-containing hybrids will contain a limited chromosomal region around the selectable marker’s integration site. It is important to note that since these fragments still contain a selectable marker, it is feasible to transfer these fragments to additional cell lines. Chromosome-mediated gene transfer (CMGT) is an-
20
1046-2023/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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MAPPING STRATEGIES WITH MICROCELL HYBRIDS
other method of introducing chromosomal fragments for regional mapping studies (8–10). In this transfection method, coprecipitates between calcium phosphate and isolated mitotic chromosomes are applied to recipient cells, and hybrids are selected by the presence of a selectable marker on the donor chromosome. This method appears to be useful as a tool for regional mapping, since cotransfer of syntenic loci occurs. However, frequent multiple interstitial events appear to contribute to chromosomal rearrangement in the introduced fragments. Porteus (9) was unable to perform any CMGT experiment that maintained the normal physical linkage of markers. He attributed this observation to deletion and religation events that occurred during the transfection process. Additionally, Pritchard and Goodfellow (10) tested the integrity of human X chromosome fragments transferred by CMGT and again observed frequent interstitial deletions. This type of chromosomal rearrangement precludes CMGT from providing adequate resources for mapping specific portions of the human genome. In contrast to fragment-containing microcell hybrids and CMGT hybrids, radiation-reduced hybrids provide a means for isolating human chromosomal fragments that lack selectable markers. These hybrids are constructed by irradiation of monochromosomal hybrid lines with subsequent fusion to a rodent ‘‘rescue’’ recipient cell line. Radiation-reduced hybrids are the only type of somatic cell hybrids that allow for high-resolution mapping of an entire chromosome. Placement of genes and markers using these types of hybrids requires extensive statistical analysis. The resolution of the maps depends on the fragmentation of chromosomes by X rays or g rays and is correlated with the physical distance (reviewed in 11–13). The basis for creating a linear order of markers along a chromosome relies on the theory that markers located close together should be rarely disrupted when the chromosome is fragmented by irradiation, while more distant markers will be more frequently disrupted. Radiation-reduced hybrid mapping, along with other somatic cell hybrid mapping methods, allows for placement of both polymorphic and monomorphic markers, unlike genetic mapping methods. Radiation-reduced hybrid maps constructed from irradiation of monochromosomal hybrid donor cells have been created for most of the human chromosomes and have provided excellent cloning resources for isolating sequences around the loci of interest (Table 1). Since this method does not rely on direct selection of the human chromosomal component in the hybrid, there is a relatively equal representation of all regions of the chromosome in the radiation-reduced hybrids; thus, maps that span the entire chromosome can be created. This chapter briefly reviews the methods used in constructing radiation-
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reduced hybrids from microcell hybrid donors for the purpose of creating high-resolution chromosome maps.
RADIATION-REDUCED HYBRIDS: BACKGROUND Goss and Harris (50, 51) performed early experiments with radiation-reduced hybrids that laid the groundwork for gene mapping with these types of hybrids. Their experiments utilized human diploid cells that were irradiated at dosages between 1000 and 4000 rads and subsequently fused to a hamster cell line deficient in the enzyme HPRT. Radiation-reduced hybrids were selected for activity of the human HPRT gene in medium containing hypoxanthine-aminopterin-thymidine (52); therefore, all the hybrids contained the portion of the human X chromosome encoding HPRT. After analyzing the hybrids for the presence of three additional X-linked genes, it was demonstrated that increasing doses of radiation greatly diminished cotransfer of other X-linked genes with the HPRT locus. These authors proposed that these genes became separated from the HPRT locus after radiation-induced chromosome fragmentation. Without selection to retain other portions of the X chromosome, these fragments would be lost as the cell population expanded. This observation was described as radiation-induced segregation of syntenic genetic loci. From these data, Goss and Harris (50, 51) determined an order for these four genes that was consistent with orders deduced from X chromosome translocations. In addition, a means to measure the relative physical distance between the markers was devised. If two loci are separated by a large distance, then it is more probable that radiation will be able to fragment the DNA between the loci and separate them. The number of events that cause segregation of one locus from another is also directly proportional to the radiation dosage. For many years the radiation-reduced hybrid methods of Goss and Harris were largely unused, partly due to the low density of markers available. By the mid 1980s, a renewed interest in these methods had developed. An important rebirth of radiation-reduced hybrid mapping appeared in 1990 (11). In this paper, a radiation-reduced hybrid panel for human chromosome 21 was constructed and analyzed with 14 chromosome 21specific markers. The creation of a map for chromosome 21 was achieved by the development of readily available computer programs that determined a map order based on the likelihood of one order versus alternative orders. An estimate of the distances between the markers was also deduced with these programs. The radiation-reduced hybrid map of chromosome 21 generated by Cox et al. (11) was compared to physical maps de-
AP: Methods
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A9(neo3/t)-5 X A9 GM7297 X14-150 Q314-2 X UrdA 9TK X GM459 HHW661 X UCW113 HD113.2B X CHTG49 HHW416 X UCW113 HHW661 X UCW113 HHW661 X UCW113 I-7 X RJK459 R21/B1 X A23 or W3GH 706-B6 clone 17 X ATS-49tg GM10156B X ATS-49tg 640-63a12 X W3GH PK87-9 X CHTG49 640-63a12 X CHTG49 762-8A X W3GH J1-11 X CHOK1 J1 X 380-6 J1 X 380-6 J1 X 380-6 J2 X 380-6 M28 X Wg3-h RJ83.1FT X RJKM PCTBA1.89 X W3gH or RAG 7AE-4 X GM459 7AE-4 X GM459 X11-4A-1d-F-e X Don/a3 20XP3542-1-4 X UV20 and 20XP3542-1-4 X Wg3h 153-E9A X adeC 153-E7BX X adeC CHG3 X GM459 CHG3 X GM459 EYEF3A6 X 380-6 C12D X W3GH GM0616 X A23 GM0616 X A23 GM7297 X B14-150
3 3(X)b 3 4 4(5)b 4 4 5(4)b 5(4)b 6 6 8 8 9 9 9 10 11 11 11 11 11 12 16 17 17 17 18 19 Hu – CH X CH Hu – CH X CH Hu – CH X CH Hu – CH X CH Hu – Ha X Ha Hu – CH X CH Hu – CH X CH Hu – CH X CH Hu – Ha X CH
Hu – M X M Hu – Ha X CH Hu – CH X C Hu – CH X CH Hu – CH X CH Hu – M X CH Hu – CH X CH Hu – CH X CH Hu – CH X CH Hu – Ha X CH Hu – Ha X CH Hu – Ha X Ha Hu – CH X Ha Hu – Ha X CH Hu – Ha X CH Hu – Ha X CH Hu – CH X CH Hu – Ha X CH Hu – Ha X Ha Hu – Ha X Ha Hu – Ha X Ha Hu – Ha X Ha Hu – M X CH Hu – Ha X Ha Hu – M X CH or M Hu – R X CH Hu – R X CH Hu – CH X CH Hu – CH X Ha
Species
3 8 – 10 8 8 8 6, 20, 50 8 8 2.5 – 25
2–8 2.2 – 25 6 6 6.5 0.5 – 8 6.5 6.5 6.5 7 40 10 5 40 1–8 4–8 50 4–8 9 9 9 9 40 7 10 8 3–6 7 5
Dose (krads)
g g X X X X g g g
X g X X g g g g g g X g g X g X X g X X X X X g X g g g g
g or X
10/18c 5 103 103 85/130c 29 67 67 86
96 86 18/72c 72/160c 109/226c 12 101/134c 109/226c 109/266c 65/93c 4/40c 11/47c 97 23 53/250c 6/47c 28 12 102 102 100 86/102 17/60c 233 38/100c 76 44/61c 98/108c 19/83c
No. RH
7 7 14 28 18 5 14 32 22
25 7 3 7 11 10 15 13 18 15 21 47 6 19 17 21 9 56 16 30 32 299 10 38 22 22 35 91 27
31 – 96a 4 – 29 11 – 22 30 – 60 22 – 28 8 – 92a 21 – 27 16 – 28 15 – 21 Up to 60 Up to 100a 9 – 82 11 – 16 4 – 48 9 – 100a 0 – 100a 4 – 54 100a 21 – 39 18 – 29 14 – 24 14 – 57 12 – 59 8 – 83 8 – 45 44 – 67 31 – 60 6 – 65 NAa NAa NAa 32 – 59 32 – 59 17 – 42 NA 22 – 70 22 – 70 4 – 29
No. loci
Retention (%)
Note. Hu, human; CH, Chinese Hamster; Ha, Hamster; M, mouse; R, rat; Hc, human chromosome; RH, radiation hybrid; NA, not available. a Hybrids selected for human DNA. b A(B), donor line carried A:B translocation, markers scored on chromosome A. c Number of hybrids analyzed/number of hybrids isolated.
21 21 21 21 22 X X X X(3)b
Parental lines
HC
Human Chromosome-Specific Radiation-Reduced Hybrid Panels
TABLE 1
44 44 11 45 46 47 48 49 15
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31, 32 33 34 35 36 37 38 39 40 41 42 43
Reference
22 JOHNSON-PAIS AND LEACH
MAPPING STRATEGIES WITH MICROCELL HYBRIDS
rived from pulsed-field gel electrophoresis. They demonstrated that the physical distances estimated by radiation hybrid mapping were proportional to the physical distances obtained from pulsed-field gels. Other intensive statistical programs are now available to enable investigators to construct linear maps from their radiation-reduced hybrid data. One of the most widely used programs is RHMAP (53), which is discussed later in the text. Since the first few mapping experiments that utilized radiation-reduced hybrids, the majority of maps created with these hybrids have been constructed by irradiating monochromosomal microcell hybrids containing a single human chromosome in a rodent cell background. These irradiated cells are subsequently fused to another rodent cell line that is deficient in an endogenous selectable gene. The major advantage of radiation-reduced hybrids constructed from monochromosomal microcell hybrids, compared to whole genome radiation hybrids constructed from diploid cells, is that they contain human chromosomal fragments derived from a single chromosome. Because of the reduced complexity, the hybrids provide a useful source of enriched human DNA for cloning experiments. Another advantage of radiation-reduced hybrids is their lack of either retention bias around a selectable marker (excluding those radiation-reduced hybrids isolated by selecting for the presence of a selectable marker) or extensive chromosomal deletion and rearrangement like that observed with CMGT hybrids (9, 10).
CONSTRUCTION OF RADIATION-REDUCED HYBRIDS Construction of radiation-reduced hybrids requires lethal irradiation of donor cells with either X rays or g rays, which induces double-strand breaks in the chromosomes. Irradiation of the cells is performed on ice so that DNA repair is minimized (50). The irradiated cells are subsequently fused to recipient cells using polyethylene glycol or inactivated Sendai virus. The recipient cells generally have a deficiency in an endogenous selectable gene that allows hybrids to be isolated in a selective medium, following the introduction of the chromosome fragment containing a normal copy of the selectable gene. When rodent 1 human monochromosomal microcell hybrids are used as the donor cell line, the normal copy of the selectable gene is usually encoded in the rodent genome. This allows for random retention of all regions of the human chromosome without preferential retention of any human locus. If a dominant selectable marker has been introduced into the human chromosome, hybrids can be isolated by selecting on this marker. However, this causes biased reten-
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tion of this region in the hybrid panel and makes it difficult to construct a map in this region (19). Choice of Parental Cell Lines The first consideration in preparing to construct radiation-reduced hybrids is the choice of cell lines for fusion partners. Most of the radiation-reduced hybrid panels appearing in the literature have been produced with hamster cell recipients (see Table 1). In addition, the majority of chromosome-specific hybrids utilized as donor cell lines contain human chromosomes in a hamster background. Irradiated human 1 hamster microcell hybrids fused to a hamster recipient are generally believed to be the best combination of fusion partners, since hamster cell lines can exist in an aneuploid state with up to four chromosome complements (32). Our laboratory has had considerable success in preparing radiation-reduced hybrid panels for human chromosomes 3 (54) and 17 (41) by fusing irradiated monochromosomal hybrids in a rat hepatoma background to the hamster cell line GM459 (NIGMS Human Genetic Mutant Cell Repository, Camden, NJ), a cell line deficient in HPRT. It is evident from other reports that not all combinations of donor and recipient cell lines produce hybrids (11, 40). It is difficult to predict whether two cell lines will be optimal fusion partners; therefore, the fusogenic properties of cells must be determined empirically. Radiation Dose The next consideration in constructing a radiationreduced hybrid panel involves the choice of dose and source of the radiation used to fragment the chromosomes. Using monochromosomal microcell hybrids as donor cell lines, it is our experience that both the length and the copy number of the chromosome in the hybrid should be considered when selecting the radiation dose. Our experience also suggests that there is much more extensive chromosomal fragmentation when equivalent doses of radiation are given from a g source compared to an X-ray source. This may be due, in part, to the difficulty of delivering accurate doses with many X irradiators. Although higher doses of radiation increase map resolution, low-retention frequencies may occur, resulting in no significant linkage between markers. Retention frequencies are calculated by counting the number of hybrids scored as positive for a particular marker divided by the total number of hybrids in the panel. Theoretically, the maximum amount of information can be obtained in a panel exhibiting a retention frequency of 50% (55, 56). The dose of radiation applied to the donor cells can be chosen to increase or decrease the number of breaks along the chromosome and between markers by using data obtained from similar radiation hybrid experiments. It has been reported that by increasing the g radiation dose from
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5000 to 25,000 rads, marker retention frequencies drop from 27 to 3%, and retained fragment sizes decrease 5- to 10-fold (15). Size of the Hybrid Panel It is also important to ascertain the number of hybrids necessary to construct a map. If the retention frequency of markers in the panel is 20–45%, then a panel of 100 hybrids is necessary to construct a linear map order (57). Sample size guidelines proposed by Lunetta and Boehnke (56) require 100 hybrids to order 16 markers with a retention frequency of 20–50% with high probability, assuming that the markers are relatively evenly spaced. If the markers are not equidistant from each other, 100 hybrids are still sufficient to order markers with 1000:1 odds, again assuming that the retention frequency is in the range of 50%. Analysis of only 50 hybrids results in lower probabilities that the predicted order is the true order. In our experience, it is best to isolate at least 130 hybrid clones from a radiation-reduced hybrid fusion. These extra clones will enable the investigator to discard hybrids that have no human DNA, those that contain an entire chromosome, or those that are duplicate clones. In this regard, it is good experimental technique to isolate only one to three hybrid clones from any one flask to eliminate the possibility of obtaining identical clones.
10. Aspirate supernatant and add 0.5 ml 50% polyethylene glycol 1500 (Boehringer Mannheim, Indianapolis, IN) prewarmed to 377C. 11. After 1 min, add serum-free medium dropwise at the rate of 1 ml for the first minute and 2 ml per minute thereafter for a total of 2 min for a final volume of 5 ml. 12. Centrifuge fused cell suspension at 1000 rpm for 5 min. Aspirate supernatant and gently resuspend fused pellet in 5 ml of prewarmed (377C) serum-free medium. 13. Incubate fused cell suspension at 377C for 90 min with occasional mixing. 14. Centrifuge cell suspension at 1000 rpm for 5 min, aspirate supernatant, and resuspend pellet in 10 ml of appropriate complete medium containing appropriate selection. 15. Plate fused cells at 1 1 106 cells per 75-cm2 flask containing complete medium plus selective reagent. Plate irradiated donor control cells in three 75-cm2 flasks at 1 1 106 cells/flask each in complete medium. Plate nonirradiated recipient cells in three 75-cm2 flasks at 1 1 106 cells/flask each in complete medium plus selective agent. 16. Replace medium in flasks 72 h after fusion and then every 5–7 days for approximately 3 weeks, after which time hybrids are clonally isolated and expanded for high-molecular-weight DNA isolation.
GENERAL PROTOCOL FOR CONSTRUCTING RADIATION-REDUCED HYBRIDS
RADIATION-REDUCED HYBRID ANALYSES
1. Expand donor and recipient cell lines to approximately 1.5 1 107 cells each. 2. Harvest donor cells and obtain cell count. 3. Aliquot 1.5 1 107 donor cells into a conical centrifuge tube. Centrifuge cells at 1000 rpm for 5 min. 4. Aspirate supernatant and resuspend cell pellet in 10 ml of serum-free medium (Dulbecco’s modified Eagle’s medium or other basic cell growth medium). 5. Place 10 ml of donor cells to be irradiated into a sterile 25-cm2 flask. If cells are to be irradiated with a g source, the cells must be placed on ice due to the length of exposure time. Irradiate the donor cells at the determined dose. 6. If using g irradiation, the recipient cells can be harvested and counted while the donor cells are being irradiated. Retain 1.5 1 107 recipient cells. 7. After irradiation of the donor cells, remove aliquots containing 1.5 1 106 cells from both the irradiated donor cells and the nonirradiated recipient cells and save for control flasks. 8. Mix 1 1 107 cells of both donor and recipient together in a sterile 15-ml conical centrifuge tube. 9. Centrifuge mixed cells at 1000 rpm for 5 min.
In most radiation-reduced hybrids, multiple human DNA fragments are integrated into rodent chromosomes, but free fragments may also be observed (12). These fragments may or may not be contiguous. Phillipe et al. (58) analyzed the rearrangement of chromosomes in radiation-reduced hybrids constructed by irradiating a human 1 hamster monochromosomal hybrid fused to a mouse recipient cell line. They detected the rejoining of human chromosomal fragments with hamster chromosomal fragments and rarely detected human fragments integrated into the recipient mouse chromosomes. They were also able to isolate small human–hamster fragments that were retained as minichromosomes. Any apparently ‘‘intact’’ hamster chromosomes probably resulted from random rearrangement of irradiated fragments. Although relatively even retention frequencies are observed for most markers along a chromosome, many radiation-reduced hybrid panels exhibit unequal retention of markers located near the centromere (reviewed in 13). Both centromeric and telomeric fragments must play an essential role in the formation of these new ‘‘chromosomes’’ from broken chromosomal fragments.
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Fluorescence in situ hybridization (FISH) analyses have proven useful for detecting the number of human fragments retained in each clone. Total human DNA is biotin-labeled by nick-translation and hybridized to metaphase chromosome spreads from each clone (59). The human chromosomal fragments are subsequently detected by an avidin–fluorescein isothiocyanate conjugate, and rodent chromosomes are counterstained with propidium iodide (Fig. 1A). Side´n et al. (15) have observed the retention of one to eight human chromosomal fragments, regardless of the applied radiation dose. Francke et al. (42) analyzed numerous radiationreduced hybrids by FISH and demonstrated that 9 of 17 hybrids exhibited clonal heterogeneity, with retention of one to five fragments of human DNA. InterAlu polymerase chain reactions (PCR) (60) can also be performed with DNA isolated from each clone, with the resulting products biotin-labeled by nick-translation (61). These PCR products can then be hybridized to normal human metaphase chromosome spreads and detected with an avidin–FITC conjugate (Fig. 1B). This type of FISH analysis, sometimes referred to as reverse chromosome painting, will provide a cytogenetic evaluation of both the number of human fragments in the clone and the chromosomal regions from which the fragments were derived. Since the human chromosomal fragments in radiation-reduced hybrids are not selectively retained and can be lost after passage in culture (12, 25), it is very important that a large quantity of DNA be isolated prior to the analysis of marker content. We have found that regrowing the radiation-reduced hybrids to obtain additional DNA results in a significant number of hybrids with altered scoring for PCR markers. These alterations can be a result of both the loss of particular sequences and the apparent acquisition of new sequences, presumably related to subpopulations of cells with differential rates of growth. Since radiation-reduced hybrids can serve as a source of region-specific sequences, segregation of the unstable fragments can be exploited in an attempt to separate the fragment of interest from the other contaminating fragments in the hybrid. By single-cell subcloning, new cell populations that contain a less complex human component can be isolated. Radiation-reduced hybrids have been used to map a variety of markers. Early studies analyzed the presence or absence of DNA markers by Southern blotting techniques (11, 14, 15, 17, 23, 30, 37, 38, 43, 45–48). More recently, PCR methods have been utilized to analyze most markers. It has been well documented that PCR is a much more sensitive detection assay than Southern analysis. Since radiation-reduced hybrids are heterogeneous populations with multiple unstable fragments, which may be lost from the clone during expansion of the cells, combining the results from anal-
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yses by Southern techniques and PCR may not be equivalent. PCR may detect a sequence that is not present in the majority of cells in the population. For example, DNA from a radiation-reduced hybrid panel for human chromosome 18, analyzed by both Southern blotting and PCR methods, demonstrated a 4% discrepancy rate in scoring between the two methods (42). The major discrepancy in scoring was observed in hybrids that were negative by Southern analysis and positive by PCR analysis. PCR also has the advantage of requiring much less DNA for analysis, which is of great benefit when confronting the expansion of a hybrid clone for large-scale DNA isolation. We perform radiation-reduced hybrid PCR analyses in 96-well microtiter plates in an Omnigene thermocycler (Hybaid Limited, Middlesex, England). Each reaction is repeated in duplicate, and the clones are scored as positive, negative, or uncertain. Any hybrid scoring that does not correlate between the two plates should be retyped, although analysis packages tolerate incomplete data. Before any markers are scored through the hybrids, the human content of each hybrid can be analyzed by inter-Alu PCR amplification (60). We have performed this type of amplification and have found hybrid clones that do not appear to contain human DNA by this type of analysis; however, when individual markers are scored, these hybrids have indeed retained human fragments. With most of our panels, we have chosen to proceed with marker analysis without scoring the hybrids for human DNA content. Only after preliminary analysis with a subset of markers are hybrids discarded based on lack of human DNA, retention of the entire chromosome, or presence of duplicated clones. After careful checking, data are entered into a spreadsheet format for analysis using the appropriate statistical packages.
STATISTICAL ANALYSES The first analysis of marker scoring should be a twopoint pair-by-pair test of each marker against all the other markers. This is used to determine linkage groups, estimate locus retention probabilities, and estimate distances between markers. These data are then analyzed by several available methods relying on multipoint statistical approaches (56). To better demonstrate the radiation-reduced hybrid mapping approach, we will briefly describe our experience with mapping the gene for glucose-dependent insulinotropic polypeptide on human chromosome 17 (62). The TWOPOINT package of Cox et al. (11) was used to generate two-point data that provided lod (logarithm of the likelihood ratio for linkage) scores, u values (frequency of breakage), and distance (D) between two
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FIG. 1. (A) Total human DNA was biotin-labeled and hybridized to a metaphase chromosome spread of a radiation-reduced hybrid constructed by fusing a human 1 rat hybrid containing human chromosome 17 with a hamster cell line. Three human fragments were observed in this hybrid. (B) Inter-Alu PCR was performed on DNA isolated from a human 1 rat 1 hamster radiation-reduced hybrid containing fragments of human chromosome 17. The PCR products were biotin-labeled and hybridized to a normal diploid human metaphase spread. One chromosome 17 fragment was observed in this hybrid.
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MAPPING STRATEGIES WITH MICROCELL HYBRIDS
markers, expressed in centirays. A distance of 1 cR6000 equals 1% of breakage between markers after exposure to 6000 rads of g radiation. With these data, pairs of markers with significant linkage were identified, and a preliminary map order was determined based on minimizing the sum of distances between markers. The next step entailed measuring the relative likelihood of one map order versus alternate orders in which the two internal markers are inverted (Fig. 2). The FOURPOINT package (11) calculates the likelihood of 12 possible orders from a set of four markers and lists the orders from most to least likely. This program provides rapid output, but is not ideal for long-range estimates of map order with more than four markers at a time, since the odds for ordering are based on internal inversions. The radiation-reduced hybrid mapping programs by Boehnke et al. (RHMAP) (53) are excellent statistical packages that combine two-point analysis with both minimum break and maximum likelihood analyses. The minimum break method uses only straightforward computations and is rapidly performed, although there are no estimates of the physical distances between markers, and it does not allow comparison of probabilities of one order versus an alternative order. Maximum likelihood provides estimates of physical distances between markers, but assumes that fragments are retained independently. These calculations are more complex and require longer computer running times. This method does allow the comparison of orders according to relative maximum likelihood. The RHMAP program can determine a map order for 40 or more markers at a time. The map is built on clusters of markers that exhibit linkage at 1000:1 odds; however, the support for order within these clusters is often not as strong. The last step in building a robust radiation-reduced hybrid map should consist of reevaluation of the marker data under the best map order based on an equal retention or centromeric retention model. Since many radiation-reduced hybrid panels demonstrate increased centromeric retention, computer simulations have been performed to compare the probability of cor-
FIG. 2. A radiation-reduced hybrid map of five loci on human chromosome 17. Distances are shown in cR6000 . Odds for order are given against the inversion of adjacent markers.
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rectly ordering markers assuming equal marker retention versus a centromeric retention model (56). The probabilities that the true order was the best order, based on equal retention or centromeric retention, were almost equal. Reevaluation of marker data should consist of examining hybrids with incomplete typing for a number of markers or a large number of breaks under the best map order. These hybrids should either be eliminated from the statistical analyses or retyped for all the markers and the data entry rechecked.
SUMMARY Methods for localizing sequences to small regions of the genome are important for increasing the resolution of physical maps of chromosomes. Radiation-reduced hybrids have provided an excellent means for creating high-resolution physical maps of markers along a chromosome. This method creates fragments that demonstrate little evidence for interstitial deletions or complex rearrangements, thus maintaining the synteny of loci along the chromosome. Radiation-reduced hybrid panels are not available for all the human chromosomes; however, human whole genome radiation-reduced hybrid mapping panels, created from diploid human fibroblast donors, have been constructed to regionally map markers from every human chromosome (63). DNA isolated from a human whole genome panel is currently available from Research Genetics (Huntsville, AL). This technology is also applicable to mapping genes and arbitrary DNA sequences in other species. In fact, radiation-reduced hybrid mapping panels have been created by investigators localizing sequences in the mouse (reviewed in 13). One disadvantage to the fragments that are created by this method is that they generally do not contain selectable markers and are not transferable to other cell lines by MMCT. However, a novel method has been introduced that allows the isolation of subchromosomal transferable fragments from any human chromosome utilizing MMCT and radiation-reduced fragmentation. In this strategy, Koi et al. (64) transfect a mammalian selectable marker into rodent cells that contain a single independently selectable human chromosome. This marked human chromosome is transferred by MMCT, which is followed by selection for both the human chromosome and the marker gene. Hybrid clones that survive the double selection will have a human chromosome tagged with both selectable markers. Microcells are prepared from these hybrids, which are subsequently irradiated and then fused to recipient cells. Hybrids that contain diverse fragments of the original human chromosome will be generated based on the various integration sites of the transfected selectable marker.
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Radiation-reduced hybrid mapping provides an integrated map for all types of markers, including arbitrary sequence-tagged sites, genetic markers, transcribed sequences, and centromeric and telomeric sequences. Markers are informative in every hybrid that is typed, and the loci do not need to be polymorphic. The amount of radiation that is used to fragment the donor chromosome(s) is a variable that can be controlled and that can be tailored to the specific experimental purposes. Radiation-reduced hybrids enable the construction of maps that bridge the gap between genetic maps and yeast artificial chromosome contig maps. With as few as 100 hybrids, data can be generated to create highresolution maps of an entire chromosome.
ACKNOWLEDGMENTS The authors thank Dr. Michael Siciliano for Fig. 1B, and Tracey B. Lewis for the data from Fig. 2. We also thank Dr. David Cox and Dr. Michael Boehnke for their radiation-reduced hybrid mapping statistical packages and Dr. Charles Leach for critical review of the manuscript.
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