GENOMICS

13, 25-34

(19%)

Characterization of Radiation/Fusion Hybrids Containing Parts of Human Chromosome i0 and Their Use in Mapping Chromosome 1 O-Specific Probes CYNTHIA B. ROTHSCHILD,*WALTER W. NoLL,t THOMAS C. GRAVIUS,$ MELISSAK. SCHUSTER,* NANCY NumE-McMErww,t CAROL JONES,!~AND DONALD W. BOWDEN**’ *Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 27157; *Collaborative Research, Inc., Waltham, Massachusetts 02154; $Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado 80262; and tDepartment of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756 Received

December30,

We have characterized a panel of somatic cell hybrid cell lines which contain different portions of human chromosome 10. GenomicDNA from the somaticcell hybrids wastested for hybridization with eachof an ordered set of probesusedpreviously to construct a genetic map of chromosome10, aswell as several additional probes, previously localized by in situ hybridization. Hybridization of an unmappedprobe to the cell line DNAs can be usedto determine its most likely position on the chromosomerelative to the mappedset of probes.Genomic DNA from two of the cell lines has beenusedto construct region-specific cosmid and bacteriophage libraries, and clones derived from these libraries were localized by hybridization to the panel of hybrid cell lines. Several of theseprobesreveal restriction fragment length polymorphisms which have been genetically mapped. Three of the probes map near the locus for multiple endocrine neoplasia type 2A, and one of these probes, BG-JC353 (DlOS167). maps between RBP3 and TB14.34 (DlOS34). Another probe, CRI-J282 (DlOS104), is closeto the FNRB locus. The panel of hybrid cell lines is thus useful for rapidly localizing unmappedprobesand asa source of DNA for the construction of recombinant libraries derived from specific regions of the chromosome. o 1992 Academic Preae, be.

INTRODUCTION Mapping of multiple endocrine neoplasia type 2A (MENBA) to human chromosome 10 (Mathew et al., 1987; Simpson et al., 1987) has stimulated several groups to construct genetic maps of the chromosome (Nakamura et al., 1988; Farrer et al., 1988; Bowden et al., 1989). These efforts were motivated, at least in part, by the desire to find additional restriction fragment length polymorphisms (RFLPs) closely linked to the disease gene. In our laboratories we have mapped MENZA to a ’ To whom

correspondence

should

1991

14-CM region bounded by the probes CRI-J170 and CRIJC145/RBP3 (No11 et al., 1988). In a report by Nakamura et al. (1989) MENBA was mapped to a ~-CM interval defined by the markers RBP3 and TB14.34. In another report the MEN2A gene was mapped to a region approximately 11 CM in length, flanked by polymorphic loci at the RBP3 locus and the FNRB locus at 10~11.2, and linked at 0.0% recombination to the polymorphic centromere locus DlOZl (Wu et al., 1990). It would be advantageous to find additional probes homologous to loci near MEN2A for additional genetic mapping studies, as markers for generation of longrange restriction enzyme maps and as markers for the initiation of chromosome walking or jumping experiments. Somatic cell hybrid cell lines have been used extensively as sources of genomic DNA enriched for particular regions of chromosomes. Development of radiation fusion hybrid technology (Goss and Harris, 1975) has made it possible for investigators to isolate cell lines containing only relatively small regions of human genomic DNA (Cox et al., 1989). We have characterized a series of radiation/fusion hybrids and somatic cell hybrids containing fragments of chromosome 10 with probes which previously had been localized by linkage mapping and/or in situ hybridization. Two of the cell lines have been used to construct both lambda phage and cosmid libraries specific for distinct regions of chromosome 10. These libraries provided a source of new chromosome 10 probes which were rapidly localized by hybridization to the hybrid cell line DNAs. Several of these new probes are derived from the pericentromeric region, mapping near the locus for MENBA. MATERIALS Hybrid cell lines. study were prepared

be addressed.

25

AND METHODS

The R88 series of hybrid by fusion with inactivated

cell lines used in this Sendai virus of y-irra-

0888.7543/92

$3.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

26

ROTHSCHILD

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TABLE

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1

Probe Summary Human library

gene mapping designation DlOZl DlOS17 D10S34 FNRB ILPR PIHB SAP1 VIM

Probe p-alpha10RP8 pMHZ15 cTB14.34 pGEM-32 pIL2R4 PA-49 SAP 28-1 hp4Fl

Localization

Source

Reference

Centromere lOpter-lop13 1OP lOp11.2 lOp14-15 lOq21.3-q23.1 lOq21-q22 lOp13

H. Willard ATCC Y. Nakamura J. Wu W. J. Leonard T. Helaakoski D. Wenger R. Baserga

Devilee et al. (1988) Lathrop et al. (1988) Nakamura et al. (1989) Wu et al. (1989) Leonard et al. (1985) Pajunen et al. (1989) Kao et al. (1987) Ferrari et al. (1987)

diated (approximately 8000 rad, cobalt-60) 762~8A cells with pro- recipient Chinese hamster ovary (CHO-B52) cells. The enzymatic block in the pro- recipient CHO-B52 cells is in the conversion of glutamate to its y-semialdehyde (Kao and Puck, 1967). The 762-8A cell line has been described previously (Fisher et al., 1987) and contains all of chromosome 10 and the Y chromosome (detectable cytogenetically). The R88 hybrid cells were selected as having retained a human gene (pro+) on chromosome 10 by propagation in Ham’s F12 medium lacking proline plus 5% dialyzed fetal calf serum. Other hybrid cell lines used in this study are 64034p61clO (derived from 762-8A), which cytogenetically contains (in addition to the Y chromosomal material) only the 1Oq portion of chromosome 10 (Fisher et al., 1987), and cell line 640-55, which contains the short arm of chromosome 10, t(lOp), chromosomes Y and 9, and additional material of unknown origin. The hybrid cell line Q314.2 contains human chromosome 3, and 665-18A is a Chinese hamster ovary cell line. Genomic DNA was isolated from the cell lines essentially as previously described (Donis-Keller et al., 1987). Library construction. A bacteriophage lambda EMBLS library was prepared using genomic DNA isolated from cell line R88-22A as previously described (Bowden et al., 1989; Donis-Keller et al., 1987). Clones from the R88-22A lambda library include probes CRI-J256 through CRI-J284. Two separate cosmid libraries were prepared using the cosmid vector c2RB (Bates and Swift, 1983) and genomic DNA isolated from cell lines R88-22A and R88-23A. Hybrid cell genomic DNA was size fractionated to approximately 40-50 kb by the combined use of Mb01 restriction endonuclease and dam methylase as described by Hoheisel et al. (1989); the DNA subsequently was treated with alkaline phosphatase and ligated to BumHI-digested c2RB, and the ligation mix packaged in vitro using a commercially available packaging extract (Stratagene). Clones containing human (as opposed to hamster) DNA inserts were identified by hybridization with labeled total human DNA as previously described (Bowden et al., 1989). Clones from the R88-22A cosmid library include probes BG-JC296 through BG-JC319, while clones from the R88-23A cosmid library include BGJC337 through BG-JC422. DNA probes. The isolation and genetic mapping of the majority of the chromosome 10 probes used to characterize the R88 hybrid cell line have been described (Bowden et al., 1989; White et al., 1990). Several other chromosome 10 probes used in this study were isolated and characterized in other laboratories and are listed in Table 1. Additional chromosome lo-specific probes were isolated from the bacteriophage lambda and cosmid libraries (see above) prepared from R8822A and R88-23A. DNA was isolated from recombinant clones by standard procedures as has been previously described (Donis-Keller et al., 1987; Bowden et al., 1989). Radiolabeling of probe DNAs, purification and restriction enzyme digestion of genomic DNAs, agarose gel electrophoresis, Southern blot transfers, and hybridizations were carried out as described by DonisKeller et al. (1987) and Schumm et al. (1988). The polymorphic chromosome 10 centromere probe, DlOZl, was hybridized using stringent conditions (50% formamide) essentially as described by Wu and Kidd (1990).

Probe localization and data analysis. Analysis of the pattern of hybridization of probes to the hybrid cell lines was carried out using the two-point algorithm derived by Cox et al. (1990). This analysis calculates the frequency with which each of two probes (markers) is retained in each hybrid cell line. Assuming that breakage between two markers is independent of marker retention, and that retention of any one fragment is independent of the retention of any other fragment, the frequency of breakage between any two markers (19, with values ranging from 0 to 1) and a corresponding lod score (logarithm of the likelihood ratio that the two probes are linked as opposed to unlinked) are calculated. Probes were also screened for their ability to reveal RFLPs with the restriction endonucleases BamHI, BglII, EcoRI, HincII, HindIII, MspI, PstI, Rsal, and TaoI as described (Donis-Keller et al., 1987). RFLPs revealed by the probes were mapped by linkage analysis relative to our set of mapped chromosome 10 probes (Bowden et al., 1989) using three-generation families from the Centre d’Etude du Polymorphism Humain (CEPH) as described (Bowden et al., 1989). Linkage analysis was carried out using the CRI-MAP (v. 2.4) program (DonisKeller, 1989) as described by Bowden et al. (1989).

RESULTS

Characterization of the R88 Hybrid Cell Lines with a Mapped Set of Probes Initially, the R88 series of hybrid cell lines was characterized by hybridization with a set of 20 probes which, by linkage analysis, were known to be uniquely ordered on chromosome 10 (Bowden et al., 1989). Genomic DNA isolated from each hybrid cell line was digested to completion with EcoRI, and electrophoresis carried out on 0.8% agarose gels from which Southern blots were prepared. In addition to genomic DNA from the R88 hybrid cell lines, several control DNAs (described under Materials and Methods) were included on each Southern blot. Control DNAs were total human genomic DNA, 7628A DNA (the parent chromosome 10 hybrid cell line), 64034p61clO DNA (containing lOq), 640-55 DNA (containing lop), CHO-B52 and 66518A DNA (hamster), and Q314.1 DNA (containing human chromosome 3). Figure 1 shows an autoradiogram resulting from the hybridization of one of these probes, CRI-J179, with such a Southern blot. Homology of the probe to chromosome 10 sequences was clear from its hybridization to genomic DNA from hybrids containing chromosome 10 fragments, and the absence of hybridization to genomic

CHROMOSOME

10 HYBRID

CELL

LINES

27

included in this analysis since its localization to 10~11.2 is well established by both linkage studies and in situ hybridization (Wu et al., 1990). It can be seen (Table 2) that the two-point lod scores derived for markers known to be adjacent are low (often ~3.0). This is a reflection of the number of radiation hybrids used in the analysis. Thus, with 18 hybrid cell lines a discordancy for two markers in only one cell line results in a lod score < 4.0 (e.g., CRI-J127 and CRIJC143), whereas discordancies in only two cell lines lower the two-point lod score to (3.0 (e.g., CRI-J170 and FNRB). In general, the two-point lod score was >2.5 when the genetic distance (Bowden et al., 1989) was less than about 5 CM. Clearly, however, there are several regions (JD12-J93, FNRB-JC145, L647-5128, JM14JC144-J90-L368, J198-VTR4) where the hybrid cell lines do not detect linkage between markers, andambiguous placement of markers derived from these regions might be expected. Each of these intervals is >lO CM as defined by genetic mapping (Bowden et al., 1989). FIG. 1. Autoradiogram of the hybridization of probe CRI-J179 to a Southern blot carrying hybrid cell line and other genomic DNAs. Each lane contains genomic DNA digested with EcoRI restriction endonuclease. The source of the genomic DNA in each lane is listed at the top. The origin or chromosome complement of the genomic DNAs is described under Materials and Methods. The marker is bacteriophage lambda DNA digested with Hind111 restriction enzyme. “Genomic” DNA is human genomic DNA.

DNA from sources which do not contain chromosome 10. Consistent with genetic mapping, the probe can be assigned to 1Oq by its hybridization to genomic DNA from 64034p61clO but not to DNA from 640-55. Each chromosome lo-specific probe hybridized to human genomic DNA and DNA from the parent cell line 762-SA (containing all of chromosome lo), but did not hybridize to CHO-B52,665-18A, or Q314.2 DNA. The hybridization of each of our mapped markers to the hybrid cell lines was analyzed using a two-point algorithm derived by Cox et al. (1990). The genetic (and in some cases) in situ localizations for each of these markers is illustrated in Fig. 2A. The average spacing between the markers is 7.7 CM (sex-equal genetic distance). The radiation hybrid two-point analysis (Table 2) calculates the frequency with which each of two probes (markers) is retained in each hybrid cell line. The further apart two markers are on the chromosome, the more likely it is that a given dose of X rays will break the chromosome between them. Assuming that retention of any one fragment is independent of the retention of any other fragment, the frequency of breakage between any two markers (8) and a corresponding lod score (logarithm of the likelihood ratio for linkage) can be calculated. Inasmuch as we are dealing with a set of loci which already have been genetically and/or physically localized, the data in Table 2 only compare values for markers which were known to be adjacent. Although FNRB is not one of our original mapped probes, it is

Characterization of Additional Mapped Chromosome 10 Loci Using the Hybrid Cell Panel Clearly, a panel of 18 hybrid cell lines is not by itself sufficient to generate a map of the chromosome. However, we were interested primarily in determining whether the R88 hybrid cell lines could be used as a tool for the rapid localization of new probes. Probes localized to a region of interest could then be pursued, for example, by genetic mapping and/or in situ localization. As a first step in our evaluation of the R88 hybrids, several probes which had been previously localized by in situ hybridization (Table 1) were mapped (using the twopoint analysis described above) using the hybrid cell lines. The results are summarized in Fig. 2B. SAP1 (sphingomyelin-activating protein) and P4HB (fi subunit of prolyl 4-hydroxylase) both have two-point lod scores > 3 with CRI-JC109, CRI-J127, and CRI-JM14, and thus cannot be unambiguously localized. VIM (vimentin) and DlOS17 are linked to CRI-J93 with lod scores of 3.66 and 2.65, respectively. For ILR2 (interleukin-2 receptor) a maximum two-point lod score of 2.0 was found with CRI-JD12. In general, these results increased our level of confidence in the utility of the R88 hybrid cell lines as a tool for the regional localization of probes since the linkage detected with the hybrids agreed with previous cytogenetic and/or genetic linkage localizations (Fig. 2A). Hybridization of the MEN2A-Linked Markers FNRB, TB14.34(DlOS34), and DlOZl to th.e Hybrid Panel We were particularly interested in mapping probes linked to the MEN 2A locus using the hybrid cell lines. As shown in Table 2, FNRB (/3 subunit of the fibronectin receptor) maps close to CRI-J170. When the data were analyzed using a four-point algorithm (Cox et al., 1990), which provides a measure of the relative likelihood for

28

ROTHSCHILD

ET

AL.

A. GENETIC

NEW

MAP

CYTGGENEW MAP

PHYSICALLY LGCALIPD

GENETlC MAP

PROBES

n ;x:‘,:

JD12 JZ62

593 5170

mJti6

FNRB

EdJc352 m

J2!FJZS8 JZSD

1

JC145 RBP3 L647 5128 5127 JC143 JC109 L1083 597 JM14

7 SAP1 P4HE

JC343 JC348 :% JC401 JC410 JC417 JC420

JCl44 J90 L368 J179 5137 J198 H

:I0

~1

VTR4

1 a

PERICENTRIIC REGION

FIG. 2. Linkage of probes to genetically mapped chromosome 10 markers by radiation hybrid mapping. (A) Genetic map of the ordered set of markers (Bowden et al., 1989) used to characterize the hybrid cell lines, as well as the cytogenetic map of chromosome 10 with the localization of DNA probes (in boxes) to specific bands as determined by in situ hybridization techniques or other physical methods. (B) Hybridization data for probes previously localized by in situ techniques or new chromosome 10 probes were analyzed for potential linkage to the ordered set of genetically mapped markers using the radiation hybrid two-point analysis (Cox et al., 1990) as discussed in the text. Solid squares indicate two-point lod scores 3 3.0, and cross-hatched squares indicate two-point lod scores 3 2.0 but < 3.0, with the corresponding mapped markers. Probes with the same pattern of hybridization to the hybrid cell lines (i.e., 8 = 0) are grouped together. Four of these probes (CRI-J258, CRI-J280, CRI-J282, and BG-JC353) which identify RFLPs were mapped and are also positioned on the genetic map (A). CRI- and BGprefixes have been omitted from the probes for clarity.

any particular order of four markers, it was found that FNRB cannot be placed on either side of CRI-J170 without ambiguity. Thus, the order J93-J170-FNRBJC145 is only 11 times more likely than the order J93FNRB-J170-JC145. Additional information collected with a newly isolated probe, CRI-J282 (see below), indicates that FNRB and CRI-J170 are close together. DlOS34 (TB14.34) has been genetically mapped to the interval between RBP3/CRI-JC145 and CRI-J170 (White et al., 1990; D.B., unpublished) and physically localized to the proximal part of lop (Nakamura et al., 1988). Mapping studies (Wu et al., 1989,199O; C.R., unpublished) also strongly suggest that the polymorphic centromere locus DlOZl (Devilee et al., 1988; Wu and Kidd, 1990) lies between the RBP3 and CRI-J170 loci. However, the location of these two probes could not be deduced by hybridization to the R88 hybrid cell lines. Upon comparison to our mapped set of probes the maximum two-point lod scores were 2.39 for TB14.34 and CRI-JM14 and 2.44 for DlOZl and CRI-L1083, whereas linkage to our MEN2A flanking markers CRI-J170, FNRB, and CRI-JC145 was not detected (i.e., two-point Iod scores < 2.0). These results are consistent with the

two-point analysis using our mapped probes in which the two-point lod score for FNRB and CRI-JC145 was only 0.03. Use of the Hybrid Panel to Localize New Chromosome 10 Markers To determine if hybridization to the R88 hybrid cell line could be used to determine the location of new chromosome 10 probes, 45 new chromosome 10 probes were hybridized to the R88 hybrids. These probes were derived from a bacteriophage lambda EMBL3 library constructed with genomic DNA from cell line R88-22A (including probes CRI-J256 to CRI-J284) and two cosmid libraries constructed from R88-22A (probes BG-JC297 to BG-JC319) and R88-23A (probes BG-JC337 to BGJC422). Cell line R88-22A DNA hybridizes to the mapped markers spanning the region from CRI-JD12 to FNRB on the p arm, but only to two markers on 1Oq (CRI-JC144 and CRI-J179), whereas R88-23A DNA hybridizes to markers spanning the region from CRI-JD12 to CRI-JC145 as well as to markers spanning the region from CRI-J128 to CRI-JC144. We chose to construct a

CHROMOSOME

10 HYBRID

CELL

29

LINES

TABLE 2 Two-Point Marker

Analysis of MaDDed Chromosome 10 Markers” Clones

observedb RH

A JD12 593 5170 FNRB JC145 RBP3 L647 5125 5127 JC143 JC109 L1053 597 JM14 JC144 J90 L365 5179 5137 5195

B 593 5170 FNRB JC14.5 RBP3 L647 5125 5127 JC143 JC109 L1053 597 JM14 JC144 J90 L365 5179 5137 J195 VTR4

++ 5 6 6 2 3 4 2 3 5 5 6 4 3 5 7 6 9 9 10 6

+4 2 2 4 2 0 2 1 0 1 0 4 1 0 7 1 1 1 0 4

-+ 3 2 0 3 1 0 2 2 1 1 3 0 1 9 0 4 1 1 0 1

Total 6 5 10 9 12 14 12 12 12 11 5 5 12 4 4 7 7 7 5 7

15 15 15 15 15 15 15 15 15 15 17 16 17 15 15 15 15 15 15 15

8’

LODd

0.75 0.45 0.23 0.91 0.44 0 0.64 0.44 0.13 0.25 0.35 0.49 0.31 0.50 0.69 0.54 0.23 0.23 0 0.54

0.20 1.24 2.60 0.03 1.12 0.44 1.12 3.20 2.31 1.53 1.10 1.55 0.25 0.50 0.59 2.65 2.65 0.59

map unitse (&cm) 150 60 26 244 55 0 103 55 14 29 42 66 37 162 115 75 25 25 0 75

‘Two-point analysis was done using the computer program developed by Cox et al. (1990) on markers (A, B) known to be adjacent on chromosome 10. CRI- prefixes are omitted from markers for clarity. b For each marker pair, the number of radiation hybrids that retain both marker A and marker B (++), only marker A (+-), only marker B (-+), or neither (-) is given. ’ 8 is defined as the frequency of breakage between the two markers A and B. d LOD is the logarithm of the likelihood ratio that a pair of markers is linked as opposed to unlinked. A numerical value cannot be calculated when 8 = 0. e Distance between markers A and B in centiRays (CR), where 1 CR, corresponds to 1% frequency of breakage between the markers upon exposure to 5000 rad of X rays.

library using R88-23A since it is the only cell line that hybridizes to the known MEN2A flanking markers CRI5170, FNRB, and CRI-JC145 (as well as to TB14.34 and the centromere probe DlOZl). Human DNA clones from each of these libraries were radiolabeled and hybridized to Southern blots of the panel of somatic cell hybrid genomic DNAs as described above. The hybridization data for each of the new chromosome 10 probes were analyzed for potential linkage to our genetically mapped probes using the two-point analysis described above. A two-point lod score > 3.0 was taken as evidence of linkage except for those probes for which additional characterization (e.g., in situ hybridization and/or genetic mapping, see below) supported the localization by radiation hybrid analysis. These data are summarized in Fig. 2B, with probes which have the same pattern of hybridization (i.e., 0 = 0) grouped together. Of the 45 new probes tested, 18 had two-point lod scores > 3.0 with one of our genetically mapped markers. For example, probes JC313,5271, and 5268, which had identical patterns of hybridization to the R88 cell line DNAs, had a maximum two-point lod score of 3.48 (0 = 0.12) with FNRB, whereas for 5256 the maximum twopoint lod score was with CRI-J170 (LOD = 3.75, 0 = 0.11). The localization of JC354 (two-point LOD = 3.2,B

= 0.13 with CRI-JM14) has been corroborated by fluorescence in situ hybridization to metaphase chromosomes (not shown). Genetic mapping of RFLPs associated with 5282 (see below) and a dinucleotide repeat polymorphism associated with JC313 (M.S. and C.R., unpublished) verified linkage of these two probes to FNRB. There was a group of eight probes (i.e., JC345JC420; Fig. 2B) which had two-point lod scores > 3.0 with CRI-J127, CRI-JC109, and CRI-JM14. This ambiguity is a reflection of the fact that the patterns of hybridization for the pairs of markers CRI-J127/CRIJC109, CRI-JClOS/CRI-JM14, and CRI-J127/CRIJM14 to the R88 hybrid cell lines are quite similar with two-point lod scores > 2.0, even though, by genetic mapping, they are only distantly linked. Sixteen of the forty-five new probes tested had twopoint lod scores between 2.0 and 3.0 with one of our mapped markers. Of these 16, the 5 probes shown in Fig. 2 have been characterized by additional studies. Using fluorescence in situ hybridization (data not shown) to metaphase chromosomes, it was found that JC353 mapped on the p arm very near the centromere, JC352 mapped to the middle of the p arm, and JC421 was mapped to the mid-q arm, thus verifying the placements suggested by the hybrid cell panel. In addition, genetic

30

ROTHSCHILD

TABLE

Marker Mapped

probes

In situ probes

New probes

(RSS-22A)

JD12 593 5170 FNRB JC145 RBP3 L647 5128 5127 JC143 JC109 L1083 597 JM14 JC144 J90 L368 5179 5137 VTR4 SAP1 P4HB VIM DlOS17 IRL2 TB14.34 DlOZl 5256 5258 5259 5260 5261 5262 5263 5267 5268

Fraction retaining

of clones marker 0.50 0.44 0.44 0.33 0.28 0.22 0.22 0.22 0.28 0.33 0.33 0.53 0.24 0.28 0.78 0.39 0.56 0.56 0.56 0.39 0.33 0.33 0.39 0.44 0.33 0.39 0.67 0.50 0.47 0.56 0.47 0.33 0.39 0.28 0.47 0.41

ET

AL.

3

Number of cell lines tested 18 18 18 18 18 18 18 18 18 18 18 17 17 18 18 18 18 18 18 18 18 18 18 18 15 18 18 18 17 18 17 15 18 18 17 17

Marker New probes continued

(RSS-22A)

New probes

(RSS-23A)

J271 5278 5282 5284 JC296 JC297 JC308 JC313 JC319 JC337 JC338 JC345 JC348 JC352 JC353 JC354 JC356 JC359 JC361 JC365 JC369 JC370 JC371 JC379 JC383 JC384 JC388 JC391 JC397 JC401 JC410 JC417 JC419 JC420 JC421 JC422

Fraction retaining

of clones marker 0.39 0.44 0.33 0.44 0.33 0.44 0.50 0.39 0.47 0.28 0.33 0.33 0.33 0.44 0.44 0.33 0.33 0.56 0.50 0.29 0.53 0.44 0.39 0.33 0.33 0.33 0.67 0.44 0.39 0.33 0.33 0.33 0.39 0.33 0.39 0.39

Number of cell lines tested 18 18 18 18 18 18 18 18 17 18 18 18 18 18 18 18 18 18 18 17 17 18 18 18 18 18 18 18 18 18 18 18 18 18 18 18

mapping of RFLPs associated with 5258 and JC353 (see below) support the placements suggested for these probes. However, genetic mapping of a RFLP associated with 5280 localized this probe to a different region of the chromosome than that suggested by the hybrid cell panel data.

fragment(s) carrying the pro- complementing activity. Upon hybridization to total human DNA, these three cell lines do appear to contain human DNA. Although the marker CRI-JC144 was retained most frequently (78%), suggesting that the pro- complementing activity is located nearby, none of the new probes isolated from RBB-22A and RBB-23A mapped to this region.

Retention Frequency for Markers Tested with the R88 Hybrid Panel

Genetic Linkage Mapping of New Chromosome 10 Probes

Although the R88 series of hybrid cell lines was selected as having a pro- complementing activity (Jones, 1975), there was a broad range of retention frequencies, ranging from 22 to 78% for the markers tested (Table 3). There was no obvious bias in the retention frequencies of probes isolated from either the RBB-22A libraries or the RBB-23A library, although as might be expected, the range of retention frequencies was narrower. None of the probes hybridized to cell lines RBB-18A and RBBlBC, and only one probe (CRI-JC145) hybridized faintly to RBB-18B. RBB-18A, B, and C may be pro+ revertants or may contain a small, undetectable, chromosome 10

In addition to hybridization to the hybrid panel DNAs, probes were tested for their ability to reveal RFLPs. Of the 45 new probes tested, 16 reveal RFLPs with at least one enzyme. The RFLPs revealed by four of these probes (CRI-J258, CRI-J280, CRI-J282, and BGJC353) have been further characterized (Table 4) and genetically mapped relative to loci constituting our earlier map (Bowden et al., 1989). Although not part of our earlier map, DlOS34 (TB14.34) was included in the analysis since it is known to be tightly linked to the MEN 2A locus. Results of conventional two-point genetic linkage analysis are shown in Table 5 and results of the multipoint linkage analysis are shown in Table 6.

CHROMOSOME

10 HYBRID

TABLE

CELL

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New RFLP Probes Constant

fragments (kb)

Probe

Locus

Enzyme

CRI-J258

DlOS93

TuqI

-

CRI-J280

DlOSlOO

PstI

-

CRI-J282

DlOS104

B&II

2.5

BG-JC353

DlOS167

RsaI

-

n Polymorphism

information

Allelic

fragments (kb) 8.0 6.0 5.8 11.0 6.8 11.0 8.0, 3.4 2.7 2.4 2.45

PIG” heterozygosity 0.53/0.59

0.33/0.38 0.29/0.41 0.29/0.40

content.

For CRI-J258, hybridization to the hybrid cell line DNAs resulted in a maximum two-point lod score of 2.44 (I? = 0.24) with the mapped marker CRI-J93 (Fig. 2B). The results of conventional two-point linkage analysis are consistent with close linkage to CRI-J93 (Table 5). Multipoint linkage analysis (Table 6) places CRI-J258 1.6 CM from, and distal to, CRI-J93. This placement, however, is only favored by odds of 2.88:1 over being proximal to CRI-J93. The probes CRI-J280 and CRI-J259, which had identical patterns of hybridization to the hybrid cell lines, had a radiation hybrid two-point lod score of 2.77 (0 = 0.22) with the marker CRI-J170. They were the only probes which hybridized to both 640-55 (lOq- cell line) and 64034p61clO (lop- cell line). Initially, we presumed that this result indicated the two cell lines carried overlapping, common parts of chromosome 10 DNA in the narrow region about the centromere, which is also the region to which MEN 2A maps. However, conventional two-point linkage analysis (Table 5) suggested that CRI-J280 is close to CRI-L647 and the RBP3 locus. Multipoint linkage analysis maps the RFLP detected by CRI-J280 to the region distal to CRI-L647 on lOq, 1.6 CM from CRI-L647 (see Table 6). This placement is favored with odds of greater than 1OOO:l over any other interval. The probe CRI-J282 has the same pattern of hybridization to the hybrid cell lines as the FNRB locus (i.e., 0 = 0), suggesting close proximity on the chromosome. Interestingly, conventional two-point linkage analysis with CRI-J282 and our other mapped probes (Table 5) indicates very tight linkage to CRI-J170: a lod score > 17.0 with no observed recombinants. In conjunction with the hybrid cell line data, this suggests that CRI5170, CRI-J282, and FNRB are very close together. Based on its hybridization to the hybrid cell lines, the probe BG-JC353 had a maximum two-point lod score of 2.65 (0 = 0.23) with both CRI-J93 and CRI-J170. This probe reveals a two-allele insertion/deletion-type polymorphism which was mapped using the restriction endonuclease RsaI (Table 4). Conventional two-point linkage

analysis (Table 5) indicates that BG-JC353 is closely linked to several pericentric loci, including RBP3 with a lod score > 21 and DlOS34 (TB14.34) with a lod score > 13. Multipoint linkage analysis (Table 6) places BGJC353 between RBP3 and DlOS34, 3.3 CM from RBP3 and 1.1 CM from DlOS34. This placement is only favored by odds of 89.5:1 over being distal to DlOS34. Either order, however, places BG-JC353 within the region containing the locus for MENBA. DISCUSSION

A panel of somatic cell hybrid cell lines containing fragments of human chromosome 10 was generated using the radiation/fusion methodology (Goss and Harris, 1975) and characterized by hybridization of their genomic DNAs with a set of probes for which the order on the chromosome had previously been determined by genetic mapping (Bowden et aZ., 1989). Radiation hybrid twopoint linkage analysis indicated that the R88 panel is, by itself, not robust enough to generate a map of the chromosome. However, we were interested primarily in using this panel of cell hybrids as a tool for the rapid localization of new chromosome 10 probes relative to our genetic map. A set of probes which had been previously localized by physical means was hybridized to the hybrid panel to check the consistency of the results with our mapped set of probes. The most likely placement of these loci (ILSR, FNRB, VIM, DlOS17, P4HB, and SAPl) was consistent with their position on the map as judged from in situ hybridization techniques and/or genetic mapping. Hybridization of a new chromosome 10 probe to the panel provides a rapid means to regionally localize the new probe relative to our genetic map. With the hybrid panels (ideally) only one hybridization is necessary to provide a localization. This is in contrast to genetic mapping of a RFLP which requires hybridization to DNAs from up to 40 or more families. An additional advantage with the hybrid panels is the ability to localize probes which do not reveal RFLPs (or other polymorphisms such as dinucleotide repeats). If the probe, such

32

ROTHSCHILD

ET

TABLE Two-Point

Linkage

AL.

5

Analysis:

LOD > 3.5

Recombination Marker

0.001

0.01

CRI-JD12 CRI-J93” CRI-J170

-6.28 12.02 6.93

linkage

-0.41 12.77 9.72

-16.47 -12.57 10.53 12.33 -5.80

linkage

-5.68 -3.39 13.21 14.07 -0.21

-0.88 -8.07 17.12 -16.16 -6.69

linkage

2.02 -0.30 16.81 -3.46 1.06

’ Locus

-25.16 -5.96 12.62 4.23 18.35 4.53 includes

two or more

-9.46 2.74 13.35 7.97 20.97 8.31 RFLPs,

including

analysis

analysis

with

those

revealed

Analysis

with CRI-J258,

3.22 5.53 6.19

1.75 2.65 3.13

3.83 4.56 9.92 10.63 3.28

3.00 3.45 6.34 7.38 2.43

1.57 1.84 2.74 3.73 1.28

3.71 5.13 9.78 6.54 5.91

2.84 3.76 6.05 4.84 4.09

1.61 1.91 2.76 2.35 1.86

4.54 7.52 8.50 7.31 14.92 8.36

3.51 5.09 5.25 4.69 9.74 5.89

1.70 2.16 2.12 1.96 4.38 2.89

CRI-J282

BG-JC353 3.42 8.59 11.51 9.28 19.39 10.00

by cosmid

4.16 8.45 8.72

CRI-J280

4.03 5.39 13.54 6.37 6.53

0.36 7.66 12.81 9.59 21.03 10.11

TABLE Linkage

with

0.40

CRI-J258

3.12 4.29 12.92 13.33 3.40

analysis

as BG-JC337, appears to be in a location of interest, it can be used as an island for locus expansion (Bowden et al., 1988). We have used the hybrid cell panel to localize several new probes isolated from libraries constructed with genomic DNA from the R88-22A and R88-23A cell lines. Eighteen of the forty-five new probes hybridized to

Multipoint

with

0.30

0.20

4.14 11.18 10.53

3.69 4.27 15.37 4.24 5.55 Two-point

CRI-J93 CRI-J170 DlOS34 CRI-JC145 RBP3 CRI-L647

with

1.05 2.59 13.87 14.28 2.68

Two-point CRI-J258 CRI-J93 CRI-J170 RBP3 CRI-L647

analysis

3.15 12.34 10.87

Two-point CRI-J170 CRI-JC145” RBP3” CRI-L647” PLAU

0.10

0.05

Two-point

fraction

clones

isolated

with

the locus probe

(Bowden

et al., 1989).

the hybrid cell panel showed significant linkage (twopoint lod scores 2 3.0) to one of our mapped chromosome 10 markers. For 16 other new probes, linkage was suggested (i.e., two-point lod scores >, 2.0). These localizations have been checked for several probes either by in situ hybridization or by genetic mapping of DNA po6 CRI-J280,

CRI-J282,

and BG-JC353

Log Order JD12-J25825.0 JD12-

1.6

593 -J170/5282-DlOS349.5 10.3

593 -J258-J170/5282-DlOS34-

JD12-J258-

593 -J170/5282-

JC353 1.1 JC353

X353

of loci” -JC145/RBP3-L647-J280-5128-5127-JC143 3.3 6.1 1.6 12.7

likelihood* .. 14.7

-JC145/RBP3-L647-J280-5128-5127-JC143

-D10S34-JC145/RBP3-L647-J28O-Jl28-Jl27-JCl43

-833.458

1.6 -833.917 .

-835.410

’ Order of RFLP loci starting with the most distal probe on the p arm (CRI-JD12) on the left, showing the possible position of new probes, CRI-J258, CRI-J282, BJ-JC353, and CRI-J280, relative to our set of mapped probes (Bowden et al., 1989) and the MENZA flanking marker DlOS34. Three possible orders are shown. Orders which are not shown have log likelihoods < -3.0 lower. For clarity the CRI- prefixes and probes distal to JC143 on the g arm have been omitted. Beneath the probe order (with the highest likelihood) are noted the sex-average distance between markers calculated with the multipoint analysis. CRI-J170 and CRI-J282 are shown as one locus since they are not separable by recombination (see Results). RBP3 and CRI-JC145 are also shown as one locus since they are separated by only one observed crossover. b Multipoint log likelihood calculated using the CRI-MAP linkage analysis program.

CHROMOSOME

10 HYBRID

lymorphisms (RFLPs or dinucleotide repeats) associated with the probes. Except for one probe (CRI-J280, discussed below) the localizations by radiation hybrid mapping agreed with mapping by other methods. While less laborious than genetic mapping, the use of this hybrid panel does have limitations. First, it is difficult to determine whether a new probe is proximal or distal to a marker on our genetic map based only on its pattern of hybridization to the hybrid panel. This is particularly true if the probe maps very close to one of the mapped markers used in the initial characterization of the cell lines. Such was the case with CRI-J258, which by genetic mapping is close to CRI-J93. Another case was the placement of CRI-J282 and FNRB which appear to be very close to CRI-J170. This limitation is general in nature, however, since neither genetic nor hybrid cell line mapping could place the probes without ambiguity. If necessary, such loci could probably be ordered using pulsed-field gel electrophoresis. A more serious limitation is that of ambiguous or incorrect placement based on the hybrid cell line data. For several probes (e.g., SAP1 and BG-JC345), this could be understood since several hybrid cell lines had similar patterns of hybridization for the three markers CRI5127, CRI-JC109, and CRI-JM14. It seems probable that a larger collection of hybrid cell lines would enable discrimination between these three loci. Our inability to map both TB14.34 and DlOZl using the hybrid panel can be explained if the centromere sequences frequently distribute more or less independently relative to neighboring sequences with the radiation/fusion hybrid method. This pattern has been observed by other workers (Benham et al., 1989; Goodfellow et al., 1990; C. Jones, unpublished). Perhaps the most perplexing result which we obtained was with the probe CRI-J280, which by hybridization to the hybrid cell line panel was mapped near CRI-J170, but when genetically mapped was found to be near CRI-L647. This placement is difficult to rationalize with the results from hybridization to the hybrid cells. This probe is certainly a candidate for additional characterization such as in situ hybridization and/or cosmid expansion. The complexities revealed with this probe emphasize the importance of using genetic as well as physical means whenever possible in mapping new probes. This would appear to be particularly important in the initial stages of characterization of somatic cell hybrids. In conjunction with information derived from our genetic map, the panel of hybrid cell lines will be useful for derivation of region-specific libraries and the localization of new probes. This is particularly useful when trying to find probes in a specific region, such as the region containing MENBA. This was the rationale for constructing libraries from the R88-22A and R88-23A cell lines, which hybridize to markers known to flank the MENBA locus, with relatively few contributions from the remainder of the chromosome. Unfortunately, none of our cell lines were limited to the centromeric region.

CELL

33

LINES

However, several probes which by hybridization to the hybrid cell line DNAs were mapped to the centromeric region have been isolated and are candidates for RFLP linkage analysis or cosmid walking experiments. Two of the probes, CRI-J282 and BG-JC353, map near the locus for MENBA and will be useful in genetic studies of this disease. In particular, BG-JC353 has been used to design sequence-tagged sites to screen yeast artificial chromosome and cosmid libraries to expand the locus. The results with CRI-J282 and BG-J353 suggest that additional characterization of probes with similar patterns of hybridization to the hybrid cell line DNAs may identify other probes in the pericentric region linked to MENBA. For example, several of these clones (e.g., 5271, JC313, and JC319) contain tracts of dinucleotide repeats and are attractive candidates for linkage studies. Clearly, additional genetically mapped loci and additional hybrid cell lines will increase the precision with which we can localize new chromosome lo-specific probes. Progress toward this end has already been made by merging genetic data from several laboratories, including our own, to produce the first CEPH consortium map of chromosome 10 (White et al., 1990). ACKNOWLEDGEMENTS This work was supported by National Institutes of Health Grant ROl CA-46806 and Grant 9013-ARG-0409 from the North Carolina Biotechnology Center. We thank K. Hsiao for contributing to the construction and characterization of the R88-22A library.

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