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Nucleic Acids Research Pages 5544-5550  

The isolation of CpG islands from human chromosomal regions 11q13 and Xp22 by segregation of partly melted molecules
Introduction
Materials And Methods
   Isolation of P1/cosmid clones and their alignment
   Segregation of partly melted molecules
   Analysis of properties of retained fragments
Results
   The isolation of CpG island from 11q13 P1 clones
   The isolation of CpG island from Xp22 cosmid clones
Discussion
Acknowledgements
References

The isolation of CpG islands from human chromosomal regions 11q13 and Xp22 by segregation of partly melted molecules

The isolation of CpG islands from human chromosomal regions 11q13 and Xp22 by segregation of partly melted molecules

Masahiko Shiraishi*, Adam J. Oates+, Xu Li§, Fumie Hosoda1, Misao Ohki1, Tiina Alitalo2, Leonard S. Lerman3 and Takao Sekiya

Oncogene Division and 1Radiobiology Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku 104-0045, Tokyo, Japan, 2Folkhälsan Institute of Genetics, Department of Medical Genetics, University of Helsinki, Mannerheimintie 97, 00280 Helsinki, Finland and 3Department of Biology,Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA

Received October 20, 1998; Revised and Accepted November 4, 1998

DDBJ/EMBL/GenBank accession nos AB005809-AB005819, AB008793, AB012166-AB012187 and AB012249-AB012263

ABSTRACT

We isolated fragments containing parts of CpG islands from human chromosomal regions chosen for expected differences in gene density by segregation of partly melted molecules. Restriction fragments of P1 bacteriophage clones covering a region of 11q13 and those of cosmid clones derived from Xp22 were recovered from bands in denaturing gradient gels that were retained following prolonged exposure to electric field. Forty-five independent fragments derived from 11q13 and five from Xp22 were isolated. Nucleotide sequence analysis revealed that 11 of the 45 fragments from 11q13 contained CpG islands including four derived from known genes in 11q13. None of the five fragments derived from Xp22 resembled CpG islands. The number of CpG island fragments obtained was consistent with the expectation based on the number of NotI restriction endonuclease sites present at these regions. Adjustment of parameters in our quasi-theoretical approach to the rate of fragment dissociation improves the discrimination between retention and non-retention. The results support probable identification of CpG island fragments by their reduced rate of strand dissociation when retarded in a denaturing gradient gel.

INTRODUCTION

We have described a procedure for the isolation of a set of fragments from cloned genomic DNA strongly enriched for CpG island sequences (1). Because the basis of the procedure is novel, we included an effort toward a theoretical rationale (1). We have termed the procedure, SPM, segregation of partly melted molecules. The SPM procedure depends on large differences in the rate of complete strand dissociation of fragments that survive digestion with restriction endonucleases whose sites appear rarely in CpG islands at a temperature just sufficient to melt part of the molecule. Fragments of unusually slow dissociation rate can be recognized by their retention in a denaturing gradient gel through prolonged application of the electric field, and a large number of retained fragments can be isolated from a single gel.

The identification of CpG islands within cloned DNA fragments is often performed on the basis of the density of CpG dinucleotides including procedures depending on the presence and/or clustering of the recognition sites for several rare-cutting restriction endonucleases, such as BssHII, EagI and SacII (2), which are predominantly located in CpG islands. In SPM, detection of CpG islands is not dependent on the presence of specific endonuclease recognition sites, which may not necessarily be present in all CpG islands.

Since 56% of human genes are associated with CpG islands (3,4), the detection of CpG islands within anonymous DNA regions often permits the recognition of many genes. SPM can serve as a valuable tool for this purpose, and in our previous study allowed the detection of a DNA fragment containing part of the CpG island of the human prostacyclin synthase gene (5). The isolated fragment was found to correspond to the 5[prime] end of the reported cDNA together with the nucleotide sequence of upstream region. SPM also circumvents the difficulty in cloning the 5[prime] end of genes, which is often difficult using cDNA, and the laborious procedures for the isolation of promoter regions of genes located upstream of the transcription initiation site.

Our initial SPM analysis of randomly selected cosmid clones containing human genomic sequences revealed that ~50% of the retained fragments were found to be derived from CpG islands (1). In this report we have extended our analysis to gene hunting on human chromosomes, and have studied chromosomal regions 11q13 and Xp22, where we have been investigating DNA aberrations in cancer and heritable diseases (6-9). We also examined the molecular nature of retention of DNA fragments in a denaturing gradient gel by adjusting parameters in an expression that assists in discriminating retention and non-retention.

MATERIALS AND METHODS

Isolation of P1/cosmid clones and their alignment

The isolation of several P1 clones from a region on 11q13 containing the ADRBK1 and GSTP1 genes has been described (9). Briefly, five known sequence tagged sites (STSs), ADRBK1, PPP1CA, P90G9S, GSTP1 and P102F3 (9) were initially used to screen the P1 library of this region. The resulting gaps were filled by further screening the library. The isolation and alignment of cosmid clones from the Xp22 region involving the GRPR locus has been also described (7). The P1 and cosmid clone DNAs were purified by equilibrium centrifugation in CsCl-ethidium bromide gradients.

Segregation of partly melted molecules

Approximately 15 µg of P1 DNAs and 10 µg of cosmid DNAs were digested to completion with restriction endonucleases, MseI, BfaI, NlaIII and Tsp509I. SPM analysis was performed essentially as described previously (1). Briefly, ~1 µg of the digested DNA fragments was electrophoresed on an 8% polyacrylamide gel containing a linear gradient of chemical denaturant. The denaturant concentration was 9% at the top and 90% at the bottom; 100% denaturant is 7 M urea, 40% formamide in 1× TAE buffer (40 mM Tris, 20 mM NaOAc, 1 mM EDTA, pH 7.4). An electric field of 10 V/cm was applied and the electrophoresis was performed for 11 h. Throughout electrophoresis the buffer and gel were maintained at a temperature of 59.5°C. SPM analysis was repeated at least twice in the case of the digests of P1 clones to examine the reproducibility of fragment retention.

Analysis of properties of retained fragments

Subcloning and nucleotide sequence analysis of retained fragments was carried out as described previously (1). Analysis of the properties of the retained fragments as potential CpG islands was performed using European Molecular Biology Network facilities (CpG-detection of CpG rich regions in DNA sequences, http://www.no.embnet.org/cpg/cpg.html ). DNA fragments having high scores ([ge]100) were regarded as being derived from CpG islands. In the case of the entire CpG islands, the score is usually >700 (data not shown). Similarity searches were carried out initially using the BLAST program (10), and the gapped BLAST program (11) on the world wide web. Calculations on the distribution of thermal stability in DNA molecules and related matters were carried out essentially as described previously (1). Analysis of methylation status of CpG islands corresponding to the retained CpG-rich fragments were carried out by methylated DNA binding column chromatography as reported previously (12,13) with slight modifications.

RESULTS

The isolation of CpG island from 11q13 P1 clones

We prepared nine P1 clones covering an ~400 kb contiguous region containing the ADRBK1 and GSTP1 genes. The alignment of the clones is shown in Figure 1A. DNAs from these P1 clones were digested with four restriction endonucleases, and subjected to denaturing gradient gel electrophoresis (Fig. 2A).


Figure 1. The restriction map of chromosomal region 11q13 and alignment of the P1 clones (A) and that of chromosomal region Xp22 with alignment of cosmid clones (B). [lambda]3150-1 is a lambda clone which contains the nucleotide sequence flanked with cosmid clones c3150-102 and c3150-2 (7). The size of each clone and the length of overlapping region of any adjacent clones are not to scale. Dotted vertical lines in (A) indicate NotI sites partially methylated in the genomic DNA (F.Hosoda, unpublished data). GRPR1, GRPR2 and GRPR3 indicate the positions of exons 1, 2 and 3 of the GRPR gene, respectively.


Figure 2. (A) SPM analysis of the complete restriction endonuclease digests of the P1 clones. Lanes 1 and 11, HindIII digests of [lambda] DNA; lane 2, P90E12; lane 3, P102F3; lane 4, P26C12; lane 5, P56D3; lane 6, P91G8; lane 7, P90G9; lane 8, P106H6; lane 9, P124C8; lane 10, P58A5. (B) SPM analysis of the complete restriction endonuclease digests of the cosmid clones. Lanes 1 and 12, HindIII digests of [lambda] DNA; lane 2, c3150-58; lane 3, c3150-102; lane 4, c3150-2; lane 5, c3150-8; lane 6, c3150-15; lane 7, c3150-45; lane 8, c3150-31; lane 9, c3150-78; lane 10, c3150-127; lane 11, c3150-14.

If retained fragments derived from two overlapping genomic clones had the same retardation level in a denaturing gradient gel and the same relative mobility in a conventional polyacrylamide gel, they were considered to be identical. In some cases, the identical nature of these fragments was confirmed by subcloning and sequencing. Retention of some fragments was not reproducible. We speculate that these fragments are at the critical edge of dissociation under the selected experimental conditions, and a subtle variation in these conditions could greatly affect their retention. Such molecules were regarded as non-retained fragments.

In the case of P1 clone P90E12, several retained bands were observed (Fig. 2A, lane 2). However, in other independent experiments with this clone, only two fragments were reproducibly retained, RP1-3 and RP1-4. Nucleotide sequence analysis revealed that fragment RP1-4 possessed properties of a CpG island (Table 1), and did not show any significant similarity to known genes or expressed sequence tags (ESTs) using the BLAST search. Fragment RP1-3 was found to be a non-island fragment.

Besides the fragments from P90E12 described above, the numbers of retained fragments obtained from P1 clones P102F3, P26C12, P56D3, P91G8, P90G9, P106H6, P124C8 and P58A5 were 6, 6, 2, 9, 14, 5, 11 and 11, respectively (Fig. 2A). Of these, and two from clone P90E12, 45 fragments were considered to be independent. Nucleotide sequence analysis revealed that, of the retained fragments from the nine P1 clones described above, 1, 1, 1, 0, 2, 4, 1, 3 and 3, respectively, were derived from CpG islands including those from the same CpG islands. In total, the number of independent fragments having properties of CpG islands was 12. The salient features of these fragments are summarized in Table 1.

Nucleotide sequence analysis revealed that fragments RP11-4 and RP5-1 contained the reported sequence of a CpG island derived from the ADRBK1 and GSTP1 genes, respectively (Table 1). Fragment RP10-6 contained the 5[prime] end of the RAD9 gene (positions 449-527, Fig. 3A) as well as a region upstream from position 1 of the reported cDNA sequence with a feature characteristic of a CpG island (Fig. 3A, Table 1). Fragment RP8-11A contained a nucleotide sequence identical to that of exon 2 of the PPP1CA gene, and clustering of CpG dinucleotides in a possible intron region of the gene. Fragment RP7-6 was almost identical to a part of the human mRNA for DRES9 protein (accession no. X98654). Although the genomic organization of this gene is not reported, the score of this mRNA sequence for CpG islands is very high (667, positions 1-3854), and the region of identity falls in this region (positions 2335-2506). These results suggest that fragment RP7-6 is derived from a part of the CpG island of this gene. Fragment RP8-4 turned out to be a part of CpG-rich repetitive element and was not regarded as a CpG island fragment.

Table 1. Retained fragments having properties for CpG islands
Clone P1 clone Length (bp) No. of sitesa Scoreb (position) Corresponding gene or EST sequence
BstUI Group II Namec Accession no.
RP1-4 P90E12 544 8 6 714 (10-538) - -
RP5-1 P102F3, P26C12 211 1 0 54d (15-51) GSTP1 M24485
RP7-6 P91G8 403 1 0 100 (205-285) DRES9e X98654
RP7-8 P91G8 583 3 1 318 (6-444) zr21h03.r1 (H) R31638
RP8-4f P91G8, P90G9 587 6 3 273 (115-273) au95f11.x1 (H) AI015997
RP8-7 P90G9 542 3 2 179 (292-527) vh14e11.r1 (M) AA475368
RP8-10 P91G8, P90G9 633 2 0 257 (92-627) zt58c01.r1 (H) AA393987
RP8-11A P90G9 494 7 2 413 (7-440) PPP1CA S57501
RP10-6 P106H6 527 6 2 503 (81-514) RAD9 U53174
RP11-4 P124C8, P58A5 590 9 3 697 (274-585) ADRBK1 U08435
RP11-7 P124C8, P58A5 1027 3 1 154 (7-321) ya49c01.r2 (H) R16209
RP11-9 P124C8, P58A5 736 12 7 811 (10-711) - -
aBstUI (CGCG). Group II enzymes are BssHII (GCGCGC), EagI (CGGCCG) and SacII (CCGCGG) (9).
bhttp://www.no.embnet.org/cpg/cpg.html
c-, absence of identical or similar sequences in database. H and M in parentheses indicate human and mouse, respectively. In the case of EST clones, only a single representative is shown.
dAlthough the score is low, this fragment is regarded as island fragment considering identity to the known sequence.
eHomo sapiens mRNA for DRES9 protein.
fNon-island repetitive sequence.

On the basis of nucleotide sequences, the remaining six of the 11 DNA fragments derived from CpG islands did not correspond to any known genes, although similarity searches revealed that four of them were identical, or highly similar (>80%), to several reported ESTs (Table 1). For example, a part of fragment RP7-8 (positions 245-383, Fig. 3B) was identical to EST clone zr21h03.r1.


Figure 3. Database matches for fragment RP10-6 and human RAD9 cDNA (A) and fragment RP7-8 and EST clone zr21h03.r1 (B). Boxed area indicates the region of sequence identity. Only a part of RAD9 cDNA sequence and clone zr21h03.r1 sequence are shown.

Methylation status of CpG islands in the genomic DNA corresponding to some of these fragments was investigated by methylated DNA binding column chromatography (12,13). All DNA fragments investigated (those corresponding to RP1-4, RP7-8, RP8-10, RP10-6 and RP11-9) had less affinity to the column, and were eluted in lower salt fractions (data not shown), indicating that the genomic sequence corresponding to these fragments are not methylated. Furthermore, it is reported that a CpG-rich region at the 5[prime] end of the human GSTP1 gene is not methylated (14). We conclude that the fragments are derived from CpG islands.

The other 33 of the 45 retained fragments were found to lack characteristics of CpG islands, and 17 of these contained Alu repeat sequences.

The isolation of CpG island from Xp22 cosmid clones

We then analyzed 10 cosmid clone DNAs covering an ~300 kb contiguous region of Xp22 containing the GRPR gene (Fig. 1B). After electrophoresis, five retained fragments, designated R15, R45, R31, R78 and R14, were obtained from digests of cosmid clones c3150-15, -45, -31, -78 and -14, respectively (Fig. 2B). Nucleotide sequence analysis revealed that these fragments did not contain sequences from CpG islands. Fragments R15, R78 and R14 contained Alu repeated sequences, while fragments R31 and R45 were derived from single copy regions.

DISCUSSION

In this study, we have applied SPM analysis for the detection of CpG islands in P1 and cosmid clones derived from contiguous chromosomal regions on 11q13 and Xp22, respectively. Of the 45 retained fragments derived from 11q13 P1 clones, 11 (24%) had regions of CpG islands, and none of the five retained fragments from Xp22 cosmid clones was derived from CpG islands. This difference in numbers of recovered CpG island fragment can be attributed to the non-uniform distribution of CpG islands in the human genome. Fluorescent in situ suppression hybridization experiments have demonstrated that on human chromosomes, CpG islands are mainly found in R bands, and more specifically in a subband of these regions known as T bands (15), indicating that the local density of CpG island along the chromosome varies markedly. The human chromosomal regions 11q13 and Xp22 are located in R bands (T bands) and G bands, respectively. The 11q13 region has clustering of CpG islands (15) and NotI sites (9) The presence of this recognition site is often indicative of CpG islands. Therefore, it was expected that there would be many CpG islands in the 11q13 region and considerably fewer CpG islands in the Xp22 region. The results obtained in this study agreed with this expectation.

In the region of 11q13 analyzed, the ADRBK1, RAD9, GSTP1 and PPP1CA genes had been assigned previously (9,16). The ADRBK1 and GSTP1 genes are known to have CpG islands, and DNA fragments containing a part of these islands were recovered by the SPM analysis. However, it was not known whether the RAD9 and PPP1CA genes were associated with CpG islands since the genomic sequence of these genes had not been reported. In this study, we have demonstrated that both these genes are associated with CpG islands. We found that the fragments derived from CpG islands of the RAD9 and PPP1CA genes were identified in overlapping genomic clones. This observation supports the previous finding that the 3[prime] ends of these genes are in close proximity, in a tail-to-tail orientation (16).

In Table 1, we show that four of the six anonymous CpG island-containing fragments, not similar to any known genes, have significant similarity to ESTs. In Figure 3B, a comparison of the nucleotide sequence of fragment RP7-8 and that of an EST clone zr21h03.r1 is shown. 3[prime] to the region of complete identity, there is a significant similarity to a consensus sequence [(C/A)AG/GT(A/G)AGT] for a 5[prime] splice junction (17). This suggests that the region 3[prime] to the identical sequence corresponds to an intron. These EST matches and similarity searches to known cDNA or genomic sequences indicated that most of the recovered fragments containing CpG islands (9 of 11; 82%) were associated with transcribed sequences.

Recent estimates suggest that ~33% of CpG islands are associated with the presence of NotI recognition sites (2). In the 11q13 region analyzed, there are five non-methylated NotI sites (Fig. 1A), and therefore one could predict that there are ~15 CpG islands. In this study 11 CpG island fragments were isolated, suggesting that most, if not all, of the CpG islands in this chromosomal region were identified by SPM.

In the SPM analysis of the cosmid clones from the 300 kb region on Xp22, none of the retained fragment was found to be derived from CpG islands. Together with previous findings (7,8), our results strongly suggested that the 300 kb region on Xp22 does not contain any CpG islands.

We have improvized an expression to relate the dissociation rate to the base sequence of the helical section (1). RHST is a parameter which we suspect to be associated with relative helix survival time.

RHST = [Sigma]{exp([Tm(i) - Tret]/q) - 1}

where Tm is the calculated temperature for each pair at which the unimolecular probability of helicity of each pair in the stable segment falls to a particular value (see below). Tret represents the equivalent temperature at the gradient level of the retarded fragment. Since the retardation depends on the residual mobility and time after the first partial melting level, and is poorly defined in the experiments, we have used the temperature required by calculation to reduce the mobility of each fragment to ~28% for Tret. The calculated equivalent retardation temperature is defined by expectation of melting of at least a sufficient number of base pairs, the same for all fragments. The value of the constant, q, is chosen to provide the best discrimination.

We find that RHST values calculated assuming Tm at 75% helicity, 95 bp melting and q value of 1.546 can discriminate retention and non-retention better than those based on our previous assumption of 50% helicity, 121 bp melting and q value of 3.185 without exception among 35 fragments investigated previously (1). All 26 retained fragments showed RHST values [ge]3.56 × 103, while all seven non-retained ones showed RHST values [le]3.19 × 103 (Table 3).

RHST values of the 50 retained fragments obtained in this study were then calculated under new assumptions. As shown in Table 3, although six of them (RP1-3, RP5-1, RP5-6b, RP8-11B, RP11-6 and RP12-8) showed RHST values <3.19 × 103, 44 of them fulfilled the new criteria for retention. This result suggests that majority of retention of DNA fragments in our gradient gel system can be predicted by calculating their RHST values.

To validate the new assumption, we investigated RHST values for arbitrary overlapping 500 bp sections spaced at 100 bp intervals covering the entire sequences of the human HRAS and MYCN genes. The results are illustrated in Figure 4A and B, respectively.

Table 2. Recalculated RHST values
Name Accession no. Position Retention RHST (×10-3)
E1 M58602 312-1015 - 1.17
E2 M58602 4227-4552 - 0.65
H1 V00574 1-490 - 1.22
H2 V00574 1411-1873 - 1.16
H3 V00574 4724-5609 - 1.67
C1 J00120 2542-2908 - 1.81
C2 J00120 4851-5244 - 0.58
N1 Y00664 1519-1828 - 1.93
N2 Y00664 6781-7343 - 3.19
1-1 M58602 1016-1876 + 9.86
1-2 M58602 4856-5844 + 3.56
2 K00650 136-1008 + 130.98
3 V00574 491-1146 + 25.53
4 J00120 2150-2501 + 24.30
5 Y00664 6133-6455 + 67.43
6 Y00664 3720-4272 + 18.42
R1-1 D31741 1-473 + 7.31
R1-2 D31742 1-446 + 7.80
R1-3 D31743 1-636 + 24.72
R2-1 D31751 1-308 + 9.25
R11-1 D31744 1-808 + 115.17
R15-1 D31754 1-305 + 72.41
R15-2 D31746 1-316 + 149480
R15-3 D31747 1-463 + 17.44
R15-4 D31748 1-517 + 30.41
R16-1 D31749 1-321 + 24.95
R16-2 D31750 1-938 + 200.76
R20-1 D31752 1-439 + 52.96
R20-2 D31753 1-505 + 6.18
R23-1 D31754 1-291 + 183.85
R27-1 D31755 1-705 + 7.31
R27-2 D31756 1-638 + 8.30
R30-1 D31757 1-524 + 50.51
R35-1 D31758 1-400 + 201.82
R35-2 D31759 1-292 + 162.94

In the case of the HRAS gene, the predicted CpG island covers positions 23-1294 (accession no. V00574), and positions 601-1100 showed markedly high RHST value (4.27 × 104) (Fig. 4A). Melting maps for positions 601-1100 (Fig. 5B) and positions 3301-3800 (Fig. 5C) revealed the presence of a sufficient length of helical domain and long lower melting domain. This feature is characteristic of retained fragments, particularly those including a portion of a CpG island and flanking sequences. The melting map of positions 501-1000 (Fig. 5A) looks similar to that of positions 601-1100 since they have similar sequences. However, after retardation, the remaining helical part is less stable in positions 501-1000 than in positions 601-1100, as shown in the melting maps. These results support the observation that uniformly G+C-rich fragments are not isolated by this method. The RHST value of positions 5601-6100 was zero. This region is comprized of tandem repeated sequence, and has a uniform melting profile (Fig. 5D).

Table 3. RHST values of DNA fragments obtained in this study
Clone P1/cosmid Length (bp) RHST (×10-3) Accession no.
RP1-3 P90E12 666 2.79 AB012166
RP1-4 P90E12 544 8.13 AB005809
RP5-1 P102F3, P26C12 211 1.75 AB005810
RP5-2 P102F3, P26C12 298 1790.5 AB012167
RP5-3 P102F3, P26C12 314 96.49 AB012168
RP5-4 P102F3, P26C12 338 132.61 AB012169
RP5-6A P102F3, P26C12 458 5.00 AB012170
RP5-6B P102F3, P26C12 379 1.30 AB012171
RP6-1 P56D3 382 50.61 AB012172
RP6-2 P56D3 465 8.65 AB012173
RP7-1B P91G8 308 9.80 AB012174
RP7-6 P91G8 403 4.25 AB005811
RP7-7 P91G8 760 4.07 AB012175
RP7-8 P91G8 583 9.11 AB005812
RP8-1A P90G9 351 25.69 AB012176
RP8-1B P90G9 307 13.55 AB012177
RP8-2A P91G8, P90G9 540 8.19 AB012178
RP8-2B P90G9 377 22.43 AB012179
RP8-3 P91G8, P90G9 798 21.20 AB012180
RP8-4 P91G8, P90G9 587 1301.5 AB005813
RP8-6A P90G9 613 5.13 AB012181
RP8-6B P91G8, P90G9 380 5.66 AB012182
RP8-7 P90G9 542 18.00 AB005814
RP8-8 P90G9 379 7.19 AB012183
RP8-9 P90G9 616 3.30 AB012184
RP8-10 P91G8, P90G9 633 4.40 AB005815
RP8-11A P91G8 494 5.09 AB005816
RP8-11B P91G8 433 1.30 AB012185
RP10-1 P106H6, P124C8 305 226.07 AB012186
RP10-2 P106H6 510 39.96 AB012187
RP10-4 P106H6 332 127.11 AB012249
RP10-5 P106H6 512 7.93 AB012250
RP10-6 P106H6 527 7.63 AB005817
RP11-1 P58A5, P124C8 262 3392.8 AB012251
RP11-2A P58A5, P124C8 523 7.67 AB012252
RP11-2B P58A5 329 26.51 AB012253
RP11-3A P58A5, P124C8 959 12.81 AB012254
RP11-4 P58A5, P124C8 590 5528.4 AB005818
RP11-5 P58A5, P124C8 533 13.47 AB012255
RP11-6 P58A5 481 1.21 AB012256
RP11-7 P58A5, P124C8 1027 4.22 AB008793
RP11-8 P58A5, P124C8 533 4.08 AB012257
RP11-9 P58A5, P124C8 736 4.25 AB005819
RP12-3 P124C8 246 145.81 AB012258
RP12-8 P124C8 481 2.57 AB012259
R14 c3150-14 227 579.20 AB012260
R15 c3150-15 289 623.02 AB012261
R31 c3150-31 370 16.35 AB012262
R45 c3150-45 568 13.86 AB012263
R78 c3150-78 282 180.17 D37794a
aPositions 452-733.

The retained fragment isolated from the cloned HRAS gene in our previous study (1) corresponds to positions 491-1146, almost coincident with positions 601-1100 as discussed above. Positions 3301-3800 and some other fragments also showed high RHST values, but the fragment corresponding to this region was not isolated because of the presence of sites for the restriction endonucleases. The principles which underlie SPM are based on the observations that (i) many genomic fragments, usually at the edge of CpG islands, dissociate more slowly during denaturing gradient gel electrophoresis and (ii) most other slowly dissociating regions are severely fragmented by a small set of restriction endonucleases. The importance of the latter is clear in considering the peaks in Figure 4.


Figure 4. RHST values plotted for uniform, overlapping, 500 bp sections of the human HRAS (A) and MYCN (B) genes spaced at 100 bp intervals. Position corresponds to the nucleotide sequence in the database. Solid lines in the plot indicate the boundary of retention and non-retention (RHST value 3.19 × 103).


Figure 5. Melting maps for positions 501-1000 (A), 601-1100 (B), 3301-3800 (C) and 5601-6100 (D) of the human HRAS gene. Position corresponds to the nucleotide sequence in the database. The contour shows the midpoint of the melting equilibrium at 75% helicity (neglecting strand dissociation) at each base pair. Solid lines indicate the calculated temperature at which the mobility is reduced to 28% of the initial value.

We obtained similar results on the MYCN gene (Fig. 4B). Positions 3901-4401 and 6201-6700 showed high RHST values (3.23 × 105 and 7.66 × 104, respectively), and the retained fragments were obtained from the corresponding positions 3720-4272 and 6133-6455, respectively (1). The former carries the edge of the island.

A 180 kb sequence containing the human RB1 gene (accession no. L11910) is known to harbor one CpG island (positions 1472-2511). When this long sequence was segmented by recognition sites of restriction endonucleases, MseI, Tsp509I, BfaI and NlaIII, and RHST values of 16 DNA fragments longer than 300 bp were calculated, eight had the values >3.19 × 103, the highest RHST value of the non-retained fragments, and seven had values <3.19 × 103 (Table 4). RHST value of positions 14799-15319 was on the boundary. This calculation suggests that a total of eight or nine fragments are expected to be retained. Two of these fragments (positions 1169-1554 and 2063-2429) are derived from both edges of the CpG island.

Table 4. RHST values of DNA fragments derived from the human RB1 gene
Position Length (bp) CpG island RHST (×10-3)
1169-1554 386 + 52.92
2063-2429 367 + 11.96
14342-14657 316 - 15.48
14799-15319 521 - 3.23
15907-16366 460 - 4.16
16815-17119 305 - 0.28
17486-17801 316 - 0.90
18286-18590 305 - 2.30
26377-26685 309 - 6.19
35618-35933 316 - 1.44
41117-41498 382 - 2.01
91649-92099 451 - 9.46
100050-100465 416 - 6.24
123891-125576 1686 - 2.82
154633-155253 621 - 1.46
163147-163600 454 - 2.03

Our calculation demonstrates that DNA fragments containing an edge of CpG island show high RHST values and hence they are retained in a denaturing gradient gel, although DNA fragments having high RHST values are also occasionally identified in non-island regions. There are five non-methylated NotI sites, considered landmarks for CpG islands, in the 11q13 region analyzed in this study. However, 11 fragments derived from CpG islands do not contain NotI sites (data not shown). This is not unexpected because it is not the entire island but generally the edge of the island that can be isolated by the SPM method, and hence the corresponding fragment may not contain such sites.

In summary, we have isolated DNA fragments containing a portion of a CpG island derived from long unsequenced DNA segments using the method of SPM. The number of retained fragments derived from CpG islands reflected the local density of CpG islands on the specific chromosomal regions. We conclude that SPM permits the efficient isolation of CpG islands within cloned DNA fragments, and therefore facilitates rapid gene identification.

ACKNOWLEDGEMENTS

This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports, and Culture of Japan (to M.S.), by a Grant-in-Aid from the Ministry of Health and Welfare, Japan, for the second term comprehensive 10 year strategy for cancer control (to T.S.), by a Research Grant on Aging and Health and a Research Grant on Human Genome and Gene Therapy both from the Ministry of Health and Welfare, Japan (to T.S.), by European Communities grant PL950889 (to T.A.), and by a grant from the National Institutes of Health (HG00345 to L.S.L.). A.J.O. was a recipient of the YKK Zip Fasteners Research Travel Award. X.L. was a Research Fellow aided by the Japan-China Medical Association.

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*To whom correspondence should be addressed. Tel: +81 3 3542 2511; Fax: +81 3 5565 9535; Email: mshirais@ncc.go.jp
Present addresses: +Lombardi Cancer Center, Georgetown University, Washington, DC 20057, USA and §Department of Biochemistry, Nantong Medical College, Jiangsu 226001, People's Republic of China

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M. Shiraishi, Y. H. Chuu, and T. Sekiya
Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using a methylated DNA binding column and denaturing gradient gel electrophoresis
PNAS, March 16, 1999; 96(6): 2913 - 2918.
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