CHAPTER 2 MATERIALS and METHODS.
3.2 GENOMIC STRUCTURE AND SEQUENCE OF
3.2.1. Genomic Structure of the PGD Locus
Mapping studies at a high resolution were carried out at the PGD locus. These led to the identification of a CpG island which was later found to be useful in long range mapping (section 3.3.3). Also, restriction mapping of the PGD genomic clones and the identification of PGD cDNA hybridizing regions allowed the determination of exon-intron structure in these fragments (section 3.2.2.3).
Previously the 5' cDNA clone pPGDH4 was shown to hybridize to 5 EcoRl restriction fragments of 10.5, 6, 4.5 ,3.0 and 0.75 kb. Somatic cell hybrid analysis showed that only the first three are on chromosome 1 and that the 3.0 kb fragment is from an unlinked locus. A clone which contained the 10.5 kb and the 4.5 kb fragments was isolated and these were found to be contiguous by restriction mapping and hybridization to the clone pPGDH4. The 6 kb fragment from chromosome 1 had not been found. The 10.5 and 4.5 kb fragments were subcloned and denoted pPGDElO and pPGDE4 respectively (Kleyn, 1990).
Restriction fragments to which the cDNA hybridized were localized as shown (figure 11). Analysis of the 10.5 kb fragment (pPGDElO) led to the discovery of a cluster of restriction sites for infrequently cutting enzymes. Hybridization of pPGDH4 to these digests showed that the coding regions were all located to one side of this cluster of sites. This raised the possibility that this cluster of sites may represent a CpG island commonly found at the 5' end of housekeeping genes (Bird, 1986). The cluster was within a 1.4kb PvuII fragment (figure 11).
fulfilled the criteria for an authentic CpG island genomic DNA was digested with EcoRl and the set of méthylation sensitive enzymes or 'rare cutters'. After Southern transfer, the DNA was hybridized to the 1.4 kb PvuII fragment and two fragments of approximately 6.2 kb and 4.3 kb were detected (figure 12).
The similar experiment using the probe pPGDH4 in place of the PvuII fragment is more complex due to cross hybridization to the pseudogene loci. However we can still detect a fragment at about 4.3 kb derived from pPGDElO (figure 13). This demonstrated that the cluster of rare cutter sites is hypomethylated in vivo and their relative location strongly points to their presence in a CpG island. Being rich in the tetranucleotide sequence for Hpall, CpG islands are also referred to as RTF islands (Hpall tiny fragment islands). The PGD island was also found to be unique and to be cleaved by Hpall into tiny fragments (figure
14).
In addition this cluster of Hpall sites was hypomethylated in the germ line. This is shown in figure 15, where a parallel digest of lymphocyte and sperm DNA is carried out. With PvuII digestion alone, the corresponding fragment is detected in both germ hne and total human DNA. As can be seen, upon cleavage with PvuII plus either MspI or its isoschizomer Hpall, the 1.4 kb PvuII fragment is lost consistent with the presence of multiple closely-spaced unmethylated 5'-CCGG-3' sites. A very faint smear is seen at the lower level of the gel representing the RTF fraction.
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E
L
pPGDH4 hybridizing regions.
Lam bda PGD extension product hybridizing region.
300 bp PvuII F r a g m e n t BgO.8 I B
l A
Ps p V Es P V Ps M - i Sc N I Ps N No P v J ___E
N P; H I I N Ps P V __I B B I B P v 1 E- EcoRI S- Smal B- BglII X- Xhol Ps- PstI N- Narl Pv- PvuII No- Notl Bs- BssHII Sc- SacII M- MluI H- HindII K- Kpnl Ps Ps _ U L_LE
1 kb 1.4 kb PvuIIFragm ent / CpG island
L
pPGDElO X nPr.DKXFigure 11. R estriction Map of Genomic Clones pPGDElO and pPGDE4.
The CpG island identified is within the 1.4 kb PvuII fragment. The regions detected by pPGDH4- and the region detected by the lambda PGD extension product at the 5' end are also indicated.
8
f
1 0 . 5Figure 12. Hybridization of 1.4 kb PvuII Fragment to Genomic
DNA Digested with EcoRI and Rare Cutter Enzymes.
Lanes: 1 - EcoRI, 2 - EcoRI & Xhol, 3 - EcoRI & BssHII, 4 - EcoRI & SacII, 5 & 6 - EcoRI & Notl, 7 - EcoRI & Smal, 8 - EcoRI & Narl, 9 - EcoRI & Mlul. The 1.4 kb PvuII fragment used as probe in this experiment contains a cluster of rare cutter sites used in the above genomic digest. If these sites are unmethylated in genomic DNA then after cleavage and
hybridization to the fragment, two fragments should be detected. This is illustrated in the above figure. The smaller fragment at about 4.3 kb is the fragment also detected by pPGDH4. The relative size of this fragment for each enzyme allowed the accurate mapping of these sites within the CpG island. In Lane 2 the presence of the
10.5 kb fragment and the weaker signal of the 8 kb fragment shows that the Xhol site present outside the island is partially methylated. The Narl digest also shows incomplete digestion probably either because of méthylation or suboptimal conditions for digestion.
A small fragment obtained in the Smal digest indicates that two Smal sites are present in the CpG island.
EcoRI EcoRI EcoRI EcoRI BssHII SacII Mlul
Tî
3
k b 10.5 6.0 4.5 4.3 3.0\
Figure 13. Hybridization of pPGDH4 to Genomic DNA Digested
with EcoRI and Rare Cutter Enzymes.
When DNA is digested with EcoRI only, pPGDH4 detects 4
fragments. The 10. 5 and 4.5 kb are in the genomic clones jpPGDElC + pPGDE4 respectively, and the 6 kb fragment is the other expressed PGD
sequence not yet cloned. The 3.0 kb fragment is from a PGD pseudogene (Kleyn, 1990). When digested with any of the rare cutter enzymes mapped in ‘ pPGDElO , a 4.3 kb fragment should be detected instead of the 10.5 kb fragment. This is because the pPGDH4 cDNA hybridizes at the 3' side of the cluster of rare cutter sites which is in a 4.3 kb EcoRI-rare cutter fragment. In lanes 1 to 3 this fragment is present although the 10.5 kb is not lost completely probably as a result of incomplete digestion with the rare cutters. The experiment shows that the sites mapped in ^ e clone pPGDElO are unmethylated
M 1 2 3 1 2 3
kb
10. 5
HTF F r a c t i o n
Figure 14 Ëthidium Bromide Stained Gel of White Blood Cell DNA Digested with EcoRl and Isoschizomers MspI and Hpall and Autoradiograph of Hybridization to the 1.4 kb PvuH Fragment
Lanes: M - 1 kb ladder, 1 - EcoRI & Hpall, 2 - EcoRI & MspI, 3 - EcoRI. The 1.4 kb PvuII fragment contains the CpG island present in the
10.5 kb genomic clone pPGDElO ~ In lane 3 a single band is detected indicating that this is a unique sequence. In lanes 2 and 3 this band is lost indicating that the island has multiple closely spaced 5 -CCGG-3' sites cleavable by both enzymes. Hpall is a méthylation sensitive enzyme thus showing that this island is also unmethylated. Extra bands present in lane 2 are most probably from incomplete digestion by MspI resulting from suboptimal reaction conditions.
Sperm cell White blood Sperm cell
DNA cell DNA DNA
i 1 2 3 1 2 3 II 1 2 3
FT
■ « White Blood cell DNA 1 2 3 k b — 1 . 4Figure 15. Ethidium Bromide Stained Gel of Germ cell and White Blood Cell DNA Digested with PvuII and Isoschizomers MspI and Hpall and Autoradiograph of Hybridization to the 1.4 kb PvuH Fragment.
Lanes: 1 - PvuII & MspI, 2 - PvuII & Hpall, 3 - PvuII.
This experiment shows that the PGD CpG island within the 1.4 kb PvuII fragment is hypomethylated in germ cell DNA as well as in white blood cells.
The two bands in the MspI/PvuII lane for WBC DNA are most probably products of incomplete digestion resulting from suboptimal reaction conditions.