SOMATIC CELL HYRID MAPPING

A panel of 12 rodent-human somatic cell hybrids has been utilised in this study for the mapping of DNA markers to different regions of chromosome 11, and to identify particularly those markers within the 1 lql3 region to facilitate further mappmg studies of MENl. Hybrid cell lines containing fragments of human chromosome 11 on a background of rodent DNA were selected which had well characterised breakpoints involving the pericentromeric region of chromosome 11 (see Table 2.2). The panel of hybrids is schematically represented in figure 3.1 and the regions of human chromosome

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Figure 3.1. A schematic representation of the 12 somatic cell hybrids ( solid bars) with breakpoints within the pericentromeric region o f chromsome II. The breakpoints divide this region into 16 intervals (A to P) as indicated by the dashed lines.

11 present in each hybrid are shown by the solid black bars. The dashed lines indicate the relative positions of the breakpoints of each hybrid, which divide this region of chromosome 11 into 16 separate intervals (A to P) to which DNA markers can be localised. The most telomeric interval on the short arm is defined by the breakpoint of hybrid B2 in 1 Ip 13 (Jones et al., 1984) and the most telomeric interval on the long arm is defined by the lower breakpoint of hybrid Jl-4 4 in l l q l 4 (Gerhard et al., 1992).

As detailed in Table 2.2, some of the hybrids contained chromosomal fragments from additional chromosomes e.g. the hybrid 1W1LA4.9 also contains Xp (Porteous et al.,

1986). Therefore, each marker analysed had to show a positive signal with the hybrid J1 which possessed an entire chromosome 11 as its sole human component. 16 polymorphic loci, 20 non-polymorphic loci and 35 cosmid clones were localised using this hybrid panel, and mapping was achieved either by using the polymerase chain reaction (PCR), or by Southern blot hybridisation.

DNA markers localised using PCR required 500 ng DNA extracted from each hybrid cell line to serve as the DNA template for the amplification reactions with oligonucleotide prim ers specific for each DNA marker (see Appendix II). Following PCR, the amplified fragments o f DNA were resolved on a 2% agarose gel and visualised by UV transillumination. The generation of a PCR product o f the expected size using a particular hybrid, demonstrated that this hybrid contains a fragment of human chromosome 11 in which the DNA marker is present. In order to ensure that the PCR product amplified from the hybrid DNA was human-specific and not as a result o f amplification o f rodent DNA present in the hybrid cell line (Table 2.2), 250 ng genomic DNA obtained from the mouse and hamster cell lines, RAG and A23 respectively, and also human genomic DNA were PCR amplified to identify the human-specific products. For each marker, there was

either an absence of products from the rodent genomic DNA or, the products obtained were o f a different size to those generated from human genomic DNA. Therefore, it was possible to identify the human-specific PCR product with all o f the markers analysed. As detailed, the hybrids J1 and 1W1LA4.9 both contain an entire human chromosome 11 and obtaining human-specific PCR products from both of these two cell lines demonstrated that a DNA marker was specifically located on chromosome 11. Additional products obtained from the remaining 9 hybrids allowed the localisation o f each marker to a specific interval on chromosome 11.

A total o f 29 DNA markers were localised by PCR and an example o f the results obtained from these mapping studies using two DNA markers K RN l and CAPNl are shown in Figures 3.2 and 3.3 respectively. In Figure 3.2, a human-specific 228bp PCR product is obtained from human genomic DNA using K RN l primers but not from the hamster and mouse genomic DNA samples. This demonstrates that the PCR primers specifically amplify human DNA and not rodent DNA. PCR products were obtained from the cell lines J1 and 1W1LA4.9 confirming the localisation of the K RN l locus to chromosome 11. A further sublocalisation was achieved from the combined results o f the other hybrids. PCR products were obtained from the hybrids Jl-4 6 , CF37, R28-4D and B2 but not from the hybrids EJNAC, Jl-11, Jl-4 4 , MCH701.8, CF52 and R185-2C2, which maps KRNl to the specific chromosomal interval labelled N.

In Figure 3.3, the localisation of the CAPNl locus is shown. As mentioned in section 1.10.6, I investigated the human CAPNl gene as a candidate for M EN l. This gene had previously been localised to chromosome 11 (Ohno et al., 1990), and the gene product of CAPNl (calpain) could potentially be a candidate for M EN l. In order to precisely map the CAPNl gene, oligonucleotide primers were designed from the 3'-

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Figure 3.2. The results obtained using the marker KRN l on the 12 somatic cell hybrids. The hybrids from which a human-specific PCR product o f 228 bp is obtained are indicated (+/-) below the photograph. These results map KRN l to interval N {see Figure 3.1)

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Figure 3.3. The results obtained using the marker CAPNl on the 12 somatic cell hybrids. The hybrids from which a human-specific PCR product o f 371 bp is obtained are indicated (+/-) below the photograph. These results map CAPNl to interval H (see Figure 3.1).

untranslated region of the gene (see Appendix ü ) which gave a 371 bp PCR product. The coding region was not selected for PCR because the genomic structure o f the gene, and hence the intron-exon boundaries, were unknown. As shown in Figure 3.3, a PCR product is obtained from human genomic DNA but not from the rodent DNAs, and in addition, products are obtained from the chromosome 11 hybrids J1 and 1W1LA4.9 confirming the localisation to this chromosome. Products are also generated from the two hybrids B2 and R185-2C2 which map the CAPNl gene to interval H, an interval to which the M EN l gene was subsequently localised to by my studies and therefore, the localisation o f CA PN l to this interval strengthened the possibility o f this being the M EN l gene.

In some instances, polymorphic microsatellite markers were localised on the hybrid panel using the same method as that used in the family linkage studies, because it was convenient to include these PCRs with the analysis of f amily members. This method involved the radiolabelling o f one of the primers prior to PCR amplification and then resolving the radiolabelled products on a 6% polyacrylamide gel which was followed by autoradiography o f the gel to reveal the products. The results obtained using the polymorphic marker D11S971 are shown in Figure 3.4. PCR products o f varying sizes were generated due to the polymorphic nature o f the marker. PCR amplification o f human genomic DNA generated two different sized products corresponding to the two alleles from each chromosome 11 whereas, the mouse and hamster genomic DNA controls did not yield any products because the prim er sequences were human-specific. The hybrids J1 and 1W1LA4.9 each yielded one PCR product because they contain a single copy of chromosome 11. Additional products were obtained from the cell lines Jl-4 6 , CF37, R28- 4D and B2 which locahse D11S971 to interval N.

Figure 3.4. The results obtained using the polymorphic marker D11S971 on the 12 somatic cell hybrids. The products obtained with this marker can range in size from 156-168 bp. The radiolabelled products which have been resolved on a 6% polyacrylamide gel are shown in the photograph. The human genomic DNA gave two alleles whereas the hybrids which yielded human-specific PCR products show one allele due to there being only one chromosome 11 in each cell. These results map D11S971 to interval N (see Figure 3.1).

primers were unavailable. In this approach, 15 pg DNA extracted from each hybrid cell line, together with 5 pg genomic DNA from human, mouse and hamster cell lines were digested with the restriction enzyme Pstl. The resulting DNA fragments were separated on a 0.8% agarose gel and transferred on to a nylon membrane by Southem blotting. The DNA markers which were localised by this approach included the RFLP markers D11S149, D11S288, PGA and D11S146, the polymorphic minisatellite marker D11S97, the non-polymorphic markers PLCPS (cDNA probe) and ZFM l (purified PCR product), and also all o f the cosmid clones. 100 ng probe DNA was radiolabelled by the random- prim ed method and then hybridised to the membrane. Due to repetitive sequences present in the cosmid clones, these probes were preannealed with denatured human placental DNA prior to hybridisation, to prevent the probe from annealing to repetitive sequences on the membrane. The minisatellite probe pMS51, which defined the locus D11S97, required competition only. An example of the results obtained by Southem blot hybridisation is illustrated in figure 3.5 in which the cosmid clone c Ill-4 4 has been localised. Multiple bands were obtained with human genomic DNA which are absent from the rodent genomic DNA lanes, and these bands are also present in the hybrids Jl-4 6 , R185-2C2, B2, J1 and 1W1LA4.9. These results confirm the localisation of the cosmid clone to chromosome 11 and further map it to interval K.

Figure 3.6 summarises the results obtained from the somatic cell hybrid mapping studies. The 16 polymorphic loci, 20 non-polymorphic loci and 35 cosmid clones are shown in the interval to which they have been localised. O f the 16 cosmid clones provided by Y. Nakamura (prefix c C Ill-) only 12 could be localised in this study due to extreme difficulty in optimising the hybridisation conditions. In addition, the position of the distal breakpoint in hybrid R185-2C2 has not been defined with respect to CF37.

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Figure 3.5. The results obtained using the cosmid clone c C Ill-4 4 on the 12 somatic cell hybrids. Radiolabelled cosmid DNA was hybridised to Pst I digested hybrid DNA electrophoresed on a 0.8% agarose gel. A multitude o f bands were obtained from human genomic DNA (indicated by arrows) which are also observed with the hybrids Jl-46, R185-2C2, B2, J1 and 1W1LA4.9. These results map c C lll-4 4 to interval K (see Figure 3.1).

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1 ^ SEA, D11S913, PJLS970_ „ D11S97. K D11S146, INT2 L * D11S971 D11S533 NON­ POLY. LOCI CINH, SSRP1 CDS, CD20, FTH1 MDU1 0 0X 8, ZFM1, D11S1226, FAU.CAPN1, PLCp3 OOF, PP1 a HSTF1.BGL1, GSTP1, ADRBK1 KRN1 NUMA1 COSMIDS D05103, B0885 ’ho3i04, FroTiër COM 1-8, c c i n - 259, c o m -297, cCIH-231 Q0870, 00950, _A062^D0J 105_. G028, 011179 D0625, B0853, A0138, G0799, Q077, COM 1-288, E072.00I11-319, COM 1-219, E0622, cOn 1-247, H025, H0295, Q07102 El 046, FI 0149, B027, cOn 1-254 cOIH-44 c o m -356, COM 1-453 H q te r

Figure 3.6, A schematic representation of the 12 chromosome I I somatic cell hybrids illustrating the localisation of 16 polymorphic loci, 20 non- polymorphic loci and 35 cosmid clones to different intervals

(labelled A to P) within the pericentromeric region o f chromosome 11. The distal R185-2C2 breakpoint has not been positioned with respect to the CF37 breakpoint and so markers located in interval J may be in interval L (indicated by *j.

These hybrids may either overlq), or as shown in Figure 3.6, may be separated by interval L, which implies that the DNA markers localised to interval J could in fact have a more distal location. Nevertheless, these studies have defined the location, and to some extent the order, o f 71 DNA markers within the pericentromeric region o f chromosome 11 and, the M EN l candidate genes Z F M l, FAU, PLCP3 and CAPNl (see section 1.9) all map to

In document Molecular genetic analysis of chromosome 11q13; localisation of the multiple endocrine neoplasia type 1 gene (Page 116-128)