This library was constructed by Dr. Dean Nizetic as part of a collaboration with the
Human Immunogenetics Laboratory. A flow-sorted chromosome 6 was used for the
construction of the library. DNA from a lymphoblastoid cell line of normal karyotype, RPETOl, was digested, ligated into the Lorist vector and transfected into D H 5a MCR (BRL). The library was robotically arrayed (Nizetic et al., 1991) onto high density grids and represents approximately 4 fold coverage of chromosome 6. It
is part of the reference library database from the ICRF (Nizetic et al., 1991).
3.2.4. PI genomic library
The total PI library was prepared by Fiona Francis (Genome Analysis Laboratory, ICRF; Francis et al., 1994) as described in section 2.2.3. The library was robotically arrayed onto high density grids, each containing 20736 clones (Nizetic et al., 1991) and represents a 1 . 2 fold genome coverage.
3.2.5. Isolation of cosmid clones from the DMB to LMP2 region
The cosmid library isolated from YAC 11.2 was screened with various probes in order to isolate new cosmids spanning the area between the DMB and LMP2 genes. Two cosmids flanking the gap were available: HA14 and U15, both of which are part of cosmid libraries prepared by Blanck and Strominger (1988 and 1990). HA 14 contains the DMA and -B genes and U15 contains the genes LMP2y TAPI, LMP7 and
TAP2 (figure 3.4). Due to the high density of repetitive DNA on the ends of these cosmids it was difficult to isolate unique probes at their extreme ends (Beck et al.,
1992b). The complete DMB cDNA was therefore used as a probe from the
centromeric side of the gap and, since the genomic sequence of U15 was available, two oligonucleotides were used to produce a 2 kb, single-copy probe by PCR from the telomeric side of the gap. This latter probe was called G El.
The DMB cDNA and G El PCR probe were used to screen the cosmid library prepared from YAC 11.2 resulting in the isolation of 10 new cosmids. Four cosmids isolated with the DMB cDNA were shorter than cosmid HA 14. These cosmids contained no novel DNA so were not further analysed. Six cosmids were isolated with the probe G E l. These cosmids were digested with the restriction enzymes EcoRI, BamHI, Notl, Clal, StuI, HindlU and Xhol, Southern blotted, and hybridised with cDNAs corresponding to the genes LMP2, TAPI, LM P7 and TAP2. Final washing stringency was 65°C, 0.2X SSC. Comparison of the restriction digest and hybridisation patterns of the new cosmids with that of cosmid U15 enabled rough mapping of the cosmids. Five of the six cosmids encoded the genes LMP2, TAPI, LMP7 and TAP2 and gave virtually identical restriction digest patterns to that of U15. These cosmids did not contain the Notl site residing in the gap. However, one cosmid, called N3, contained the Notl site predicted to reside in the gap by physical mapping (Ragoussis et al., 1991) and extended the cloned region by 4 kb (figure 3.4).
At the same time filters from the flow-sorted chromosome 6 cosmid library
were screened with the DMB cDNA and GEl probe. Three clones were isolated using
the DMB cDNA. Hybridisation studies showed that two of these encoded the genes
DMB and DMA (centromeric of DMB). The third clone, A 12, was of interest since it showed a positive hybridisation signal with DMB yet a negative hybridisation signal with DMA indicating that it would extend furthest into the gap. From restriction enzyme mapping and comparison with cosmid HA 14, cosmid A12 extended approximately 21 kb telomeric of HA 14. On the other side of the gap eight cosmids
kb 0 20 40 60 80 100 120 DM A DM B LMP2 TAPI LM P7 TAP2 J___ L Y h n T 1 1 1 S t i l l I I I 1 1 1 1 1 I I I ( +) I I I I I III 1 C l a l --- 1--- 1--- H A 14 I_________________ I A 12 U15 U l O C O SM ID CLONES A15 N3 PI CLO NE DP2
Figure 3.4. Map of the MHC class II region between the genes DMB and
LMP2. The map shows the position of cosmids and a PI clone isolated, in this study, from the cosmid library constructed from YAC 11.2 (N3), the flow sorted chromosome 6 cosmid library (A12/A15) or from the PI library (DP2).
These are shown relative to the previously described cosmids HA 14, U15 and UlO (Blanck and Strominger, 1988; Blanck and Strominger 1990). (+), a large number of StuI sites at the telomeric end of the map.
were isolated with the probe G El (figure 3.5). Seven of these cosmids were positive with the LMP2 cDNA probe and one was negative. This last cosmid, A 15, contained the Notl site and extended 30 kb into the gap. Initial restriction enzyme mapping and hybridisation studies showed an overlap between the two cosmids A12 and A15 (figure 3.6). Comparison of the restriction enzyme digest patterns of cosmids A15 and A12 using the enzymes Notl, Xhol, StuI and Clal established a 6 kb overlap between
the two cosmids. These results were confirmed by using fluorescence in situ
hybridisation (FISH) on free chromatin fibers with both cosmids detected in different colours. The FISH was carried out by Dr. G. Senger in the Human Cytogenetics Laboratory at the ICRF.
The cosmid clones A12, A15 and N3 were used for FISH on free chromatin released from peripheral blood lymphocytes of a healthy donor (Senger et al., 1993). The fluorescence signals on released chromatin obtained with each cosmid were visible as either red or green dotted lines that were partially overlapping. In figure 3.7a, cosmid A 12 is visualised in red and cosmid A15 in green, showing a 6 kb
overlap. The overlaps between cosmids A 12/HA 14 and A15/U15 were demonstrated by the same technique showing a 20 kb overlap between A 12 and HA 14 and a 10 kb overlap between A15 and U15 (figures 3.7b and 3.7c).