ANALYTICAL
BIOCHEMISTRY
190,326-330
(1990)
Monitoring Mammalian Genome Rearrangement Mid-Repetitive Sequences as Probes’ Lo-Chun
Au,* Paul
0. P. Ts’o,t*’
*Medical Research Department, The Johns Hopkins University,
Received
April
and Ming
Yit
Veterans General Hospital, Taipei, Taiwan, Republic of China, and tDivision School of Hygiene and Public Health, Baltimore, Maryland 21205
Press,
Inc.
Repetitive sequences commonly exist in the genome of eukaryotic cells (l-3). In many cases, repetitive sequence serves as the hotspots for rearrangement events via the mechanism of homologous recombination (4,5). Homologous recombination between eukaryotic repeated DNA (nonallelic) sequences generates gene conversion, reciprocal translation, deletion, and amplification (4,6). Genetic disease may result from the consequence of such recombination (7,8), so investigation has been initiated to monitor the heterogeneity and instability of the locations of repetitive sequences (9). In this paper, we have developed a procedure which can monitor the possible genome rearrangement involving ’ This work supported by the Department of Energy, FG02-88-ER60636. * To whom reprint requests should be sent. 326
of Biophysics,
20, 1990
By utilization of mid-repetitive sequences, the intracisternal A particle (IAP) gene, as a probe, genome rearrangement involving IAP genes and their neighboring sequences in rodent cells can be monitored. This is based on electrophoretic separation of the twice digested restriction fragments of genomic DNA in a 2-D pattern. The first digestion was done in solution followed by electrophoresis of the restriction fragments in the first dimension. A second restriction enzyme digestion was carried out in situ in the gel followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. After Southern blotting, the DNA on the filter is hybridized with a probe that is a fragment located near the 5’ end of the IAP gene, but does not overlap with the 5’ long terminal repeat (LTR). The exposed X-ray film revealed about 370 distinct spots in the 2-D maps. In comparing the 2-D maps, genome rearrangement involving IAP was detected. o 1990 Academic
with
Contract
DE-
the mid-repetitive sequences ( lo’-lo3 copies/genome in mammalian cells). This approach is based on the ability to separate the restriction fragments of genomic DNA in the agarose gel by a Z-D3 pattern (10). After Southern blotting, the DNA on the filter was hybridized with a labeled end fragment of a mid-repetitive sequence. Comparing the 2-D restriction fragment maps of one test cell type versus that of another reference cell type, we hope to be able to monitor any rearrangement around the mid-repetitive sequences between these two cell types in question. This method will be useful in investigating the role of DNA rearrangement in differentiation and/or carcinogenesis involving the mid-repetitive sequences. For this study we have monitored the intracisternal A particle (IAP) gene. IAP genes are endogenous retrovirus-like sequences found in rodents (11,12), with 102lo3 copies per genome. IAP genes are dispersed among all the chromosomes (13). These sequences are structurally similar to the transposons having a long terminal repeat (LTR) at each end. Some of the LTRs have transcriptional promoter and enhancer activities (14,X). Endogenous viral sequences do activate cellular genes including protooncogenes in some cases (16-B). On the basis of the reasons mentioned above, monitoring the genomic DNA rearrangement involving IAP genes would be of value. MATERIAL
AND
METHODS
Cell Cultures The cell line PC44 was provided by Dr. Yasushi Yokokawa of our division. This cell line was derived from a clone of an established diploid myoblast cell line pre3 Abbreviations used: IAP, intracisternal A particle; Z-D, mensional; BSA, bovine serum albumin; LTR, long terminal SDS, sodium dodecyl sulfate; BSA, bovine serum albumin.
two direpeat;
0003-2697/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
MID-REPETITIVE
SEQUENCES
AS PROBES
3’L 2
TR
FIG. 1. The probe and its location in the map of an IAP gene from Syrian hamster. Abbreviations: B, BglII; H, HindIII; P, PstI.
pared from g-day gestation Syrian hamster embryo cells by continuous treatment with phorbol 12,13-didecanoate at 0.1 pg/ml(l9). The PC44 cells were cultivated in ERM (Dulbecco’s Eagle reinforced medium) with 20% fetal calf serum. Another cell line, BPGT was derived from the same isogenic Syrian hamster as PC44. BPGT is a highly tumorigenic, chemically transformed clone of Syrian hamster embryo cells (20). Extraction
of High Molecular
Weight DNA
Cells from two to four T150 flasks were trypsinized and collected by centrifugation. The cells were lysed in 1 ml of cell lysis buffer (20 mM Tris, pH 8, 0.1 M NaCl, 0.5% v/v Nonidet P-40, 10% sucrose). The cell lysate was transferred to a l&ml centrifuge tube and was overlayered on 2 ml of cell lysis buffer containing 30% sucrose. Nuclei were pelleted by centrifugation (10K rpm, 10 min, in HB-4 rotor). The nuclei were lysed in 2 ml of proteinase K buffer (0.5% SDS, 10 mM EDTA, 50 mM Tris, pH 8) containing 2 mg of proteinase K. The hydrolysis was carried out at 37°C for 2.5 h. One-tenth volume of 5 M NaClO, was then added and lysate was extracted once with phenol:chloroform (1:l) and then twice with chloroform. The DNA solution was dialyzed against 1X SSC overnight. After dialysis, the DNA solution was treated with RNase A (50 pg/ml) at 37°C for 1 h. One-third volume of 4~ proteinase K buffer was added, followed by proteinase K digestion (100 pg/ml) at 37°C for 1 h. The DNA solution was extracted once with phenol:chloroform (1:l) and twice with chloroform. After the addition of l/9 vol of 3 M sodium acetate, pH 7,2 vol of 95% alcohol (-20°C) was overlayered on the DNA solution. DNA was spooled out by a glass rod. DNA was then redissolved by dialyzing against TE buffer (5 mM Tris-HCl, 1 mM EDTA, pH 8.2).
IN
GENOME
REARRANGEMENT
327
with EcoRI reaction buffer (0.1 M Tris, pH 7.7, 50 mM NaCl, 10 mM MgCl,) plus 250 pg/ml bovine serum albumin (BSA) by shaking 1 h in room temperature. Then the gel strip was put in a small column (total volume, I5 ml) which contained EcoRI reaction buffer plus 250 pg/ ml BSA and 200 units/ml EcoRI. The column was gently rotated for 20 h at 37°C. After the EcoRI in situ digestion, the gel strip was equilibrated with IX TPE buffer. The gel strip was then fused with a 1% gel slab which has been equilibrated with 3X TPE, 25% formamide, by adding melted agarose (1% in TPE) to fill up the gap. Electrophoresis was carried out at 4°C in 1X TPE buffer. After electrophoresis, the gel was stained with ethidium bromide. Southern
Blot and Probe Hybridization
The Southern blot, hybridization, and washing protocols were carried out in accordance with the previously described methods (21). The DNA fragments in the 2-D gel were transferred onto Gene Screen membrane by Southern blotting. After baking, the membrane was prehybridized in hybridization buffer (0.45 M NaCl/ 0.045 M sodium citrate/O.l% SDS/0.02% polyvinylpyrrolidone/0.02% Ficoll/O.O2M potassium phosphate, pH 6.8) containing 100 @g/ml of sheared and denatured herring sperm DNA at 61°C for 5 h. The hybridization buffer containing 1 Fg of 32P-labeled IAP probe (see below) at 61°C for 18 h. The washing procedure was carried out at 61°C. Probe The 1.9-kb IAP Hind111 fragment of Syrian hamster (Fig. 1) was provided by Dr. Anne Brown (unpublished data). The 1.9-kb IAP fragment contains the same BglII and P&I restriction sites and orders as described by Ono and Toh (22). The 350-bp BglIIIPstI fragment was isolated and labeled by nick translation. The reaction mixture contains 1 pg probe DNA, 50 I’IIM NaPi, pH 6, 10 mM MgCl,, 1 mM EDTA, 60 PM dATP and dGTP, and 50 &i [a-32P]dCTP (3000 Ci/mmol), and 25 units of DNA polymerase I (Boehringer-Mannheim Biochemicals). The mixture was incubated at room temperature for 6 min and then shifted to 13’C for 2 h. The specific activity was 3.5 X lo7 dpm/pg DNA.
2-D Agarose Gel of Genomic Restriction Fragments
RESULTS
High molecular weight DNA was digested completely by BgZII. The digested DNA was loaded in a well of a 1% agarose gel (length, 25 cm; width, 20 cm; thickness, 0.46 cm). Electrophoresis was carried out in 1X TPE buffer (0.08 M Tris-phosphate, 0.008 M EDTA, pH 7.7). After first-dimension electrophoresis, the DNA fragments in the agarose gel were digested with EcoRI by the following method: the gel strip was first equilibrated
Encouraged by the results of 2-D mapping of restriction fragments of Escherichia coli genomic DNA (lo), we tried to extend our work to the mammalian cell for creating a 2-D map of mid-repetitive sequence. High molecular weight genomic DNA from Syrian hamster was digested completely by BgZII. An ll-pg sample of digested DNA was loaded in a well of 1% agarose gel. The procedure for generating a 2-D gel map of genomic
AND
DISCUSSION
328
AU,
23 I
94 I
66 I
TS’O,
44 I
AND
YI
20 I 23
94
23 I
94 I
66 I
44 I
20 I 23
94
20 FIG. 2.
The Z-D restriction fragments maps of the IAP 5’ end containing sequences of PC44 (a), and BPGT (c). The procedure is described under Materials and Methods. The small arrows point out the difference in spots between the two maps. The arrow pointed upwards indicates the presence of a spot and that pointed downwards indicates the absence of a spot. Control for the full digestion, 1 pg of X phage DNA was electrophoresed into the gel. The piece of gel containing the DNA was cut and underwent EcoRI in-gel digestion together with the experimental gel strip. The digested fragments were revealed after electrophoresis (b).
FIG. 3. experiments.
Two
2-D restriction fragment maps of the genomic DNA PC44 cells The patterns of various clusters of spots can be clearly recognized.
with
the
IAP
5’ end
as probes
showing
reproducibility
of two
330
AU,
TS’O,
restriction fragments was followed (see Materials and Methods). After ethidium bromide staining, only a few spots were resolved (data not shown), as compared with the 2-D gel maps of E. coli genomic DNA (10). This lack of resolution was due to crowding of too many spots overlapping with each other. Only the highly repetitive sequences can be seen as distinct spots as revealed by staining. Subsequently the Syrian hamster genomic DNA fragments in the 2-D gel were transferred to membrane by Southern blotting and hybridized with IAP probe as described under Materials and Methods. The probe used was a 350-bp BgZII/PstI fragment cloned from a Syrian hamster IAP gene (Fig. 1). This probe is close to but does not overlap with the 5’ LTR, and thus, this probe can only detect the sequences flanking the 5’ LTR but cannot detect the sequences flanking the 3’ LTR. In view of the diversity of IAP genes in the genome, we selected less stringent conditions (61’C) for the probe hybridization. About 370 distinct spots were revealed after autoradiography (Fig. 2A). The difference in intensities of these spots observed in the 2-D map may be caused by differences in the extent of homogeneity between these individual IAP sequences and the probe used in this experiment. In addition, some IAP sequences may be reiterated in tandem in the genome. Sequences with a higher copy numbers will bring about higher intensity. Since the DNA was not evenly distributed in the gel, a control was carried out for every in-gel digestion in order to ensure that all the DNA fragments in the gel strip were fully digested. A l-bg sample of X phage DNA was electrophoresed into the gel. The piece of gel containing the X phage DNA was cut and underwent in-gel digestion together with the gel strip containing the hamster DNA. Since the X DNA concentration at the band is higher than hamster DNA at any place in the gel strip, the full digestion of that X band, as shown by electrophoresis (Fig. 2B), ensures the full digestion of DNA in the gel strip. The dark strip appearing at diagonal place represents the IAP-containing DNA restriction fragments which did not have EcoRI cutting site. A rearranged IAP-containing fragment might not be detected if this fragment does not have the EcoRI cutting site and thus locates at the dark strip. In this case, different restriction enzymes (for the first or second digestion) should be considered. This procedure has been shown to be highly reproducible. The PC44 genomic DNA have been analyzed four times through this 2-D gel electrophoresis procedure. The results have been remarkably similar and two of the four gel patterns are shown in Fig. 3. The genomic DNA for BPGT were also analyzed repeatedly in yielding very similar patterns. Polymorphism in DNA sequence exists in individuals of some species, so the DNA samples for the 2-D com-
AND
YI
parison should be obtained from the same animal or from isogenic animals. The question of changes in DNA methylation should also be considered. The restriction enzymes chosen for this experiment, EcoRl and BglII, are not sensitive to the methylation of -GGCCor GCGC- sequences. On the other hand, by the choice of appropriate enzymes pairs, the change of DNA methylation pattern can be the subject of the investigation instead of DNA rearrangement. In summary, a highly reproducible procedure is demonstrated here for monitoring mammalian genome rearrangement. This procedure utilizes two restriction enzyme treatments and 2-D gel electrophoresis for separation and uses the appropriate portion (ends) of a midrepetitive DNA sequence as probes in Southern blotting for detection. This procedure has the capability of monitoring up to 1000 regions of the genome for rearrangement adjacent to a chosen repetitive DNA sequence which are hotspots for such activities. REFERENCES 1. Flavell,
I.-H.
2. Doring, 127-130.
H. P., and
3. Baltimore,
(1983)
Cell 34,415-419. Tillmann,
D. (1985)
E. (1984)
Nature
(London)
30’7,
Cell 40,481-482.
4. Fasullo, M. T., and Davis, R. W. (1987) Proc. N&l. Acud. Sci. USA 84,6215-6219. 5. Kupiec, M., and Petes, T. D. (1988) Genetics 119,549-559. 6. Stark, G. R., Debatisse, Cell 5’7, 901-908.
M.,
Giulotto,
E., and Wahl,
G. M. (1989)
I. Latt, S. (1981) Annu. Reu. Genet. 15, l-57. 8. Lerman, M. A., Schneider, W. J., Sudhof, T. C., Brown, M. Coldstein, J. L., and Russell, D. W. (1985) Science 227,140-146. 9. Uitterlinden, (1989) Proc.
A. G., Slagboom, P. E., Knook, Natl. Acad. Sci. USA 86,2742-2746.
10. Yi, M., Au, L., Ichikawa, Sci. USA 87,3919-3923. 11. Calarco, 93.
N., and Ts’o,
P. G., and Szollosi,
P. (1973)
P. (1990) Nature
S.,
D. L., and Vijg, Proc. Natl. (London)
12. Chase, P. G., and Piko, L. (1973) J. N&l. Cancer Inst. 1973. 13. Lueders, K. K., and Kuff, E. L. (1977) Cell 12,965-972.
J.
Acad.
243,91-
51, 1971-
14. O’Connell, C. D., and Cohen, M. (1984) Science 226,1204-1206. 15. Kessel, M., and Khan, A. S. (1985) Mol. Cell. Biol. 5, 1335-1342. 16. Rasheed, S. (1982) Proc. Natl. Acad. Sci. USA 79, 7371-7375. 17. Dickson, C., Smith, R., Brookes, S., and Peters, G. (1984) Cell 37, 529436. 18. Chen, S. J., Holbrook, N. J., Mitchell, K. F., Vallone, C. A., Greengard, J. S., Crabtree, G. R., and Lin, Y. (1985) Proc. N&l. Acad. Sci. USA 82.7284-7288. 19. Yokogawa, Y., and Bruce, S. A. (1986) J. Cell Biol. 103,26a. [Abstract.] 20. Ts’o, P. 0. P. (1977) The Molecular Biology of the Mammalian Genetic Apparatus, North-Holland, Amsterdam. 21. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 22. Ono, M., and Toh, H. (1985) J. Viral. 66, 387-394.
BIOCHEMISTRY
190,326-330
(1990)
Monitoring Mammalian Genome Rearrangement Mid-Repetitive Sequences as Probes’ Lo-Chun
Au,* Paul
0. P. Ts’o,t*’
*Medical Research Department, The Johns Hopkins University,
Received
April
and Ming
Yit
Veterans General Hospital, Taipei, Taiwan, Republic of China, and tDivision School of Hygiene and Public Health, Baltimore, Maryland 21205
Press,
Inc.
Repetitive sequences commonly exist in the genome of eukaryotic cells (l-3). In many cases, repetitive sequence serves as the hotspots for rearrangement events via the mechanism of homologous recombination (4,5). Homologous recombination between eukaryotic repeated DNA (nonallelic) sequences generates gene conversion, reciprocal translation, deletion, and amplification (4,6). Genetic disease may result from the consequence of such recombination (7,8), so investigation has been initiated to monitor the heterogeneity and instability of the locations of repetitive sequences (9). In this paper, we have developed a procedure which can monitor the possible genome rearrangement involving ’ This work supported by the Department of Energy, FG02-88-ER60636. * To whom reprint requests should be sent. 326
of Biophysics,
20, 1990
By utilization of mid-repetitive sequences, the intracisternal A particle (IAP) gene, as a probe, genome rearrangement involving IAP genes and their neighboring sequences in rodent cells can be monitored. This is based on electrophoretic separation of the twice digested restriction fragments of genomic DNA in a 2-D pattern. The first digestion was done in solution followed by electrophoresis of the restriction fragments in the first dimension. A second restriction enzyme digestion was carried out in situ in the gel followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. After Southern blotting, the DNA on the filter is hybridized with a probe that is a fragment located near the 5’ end of the IAP gene, but does not overlap with the 5’ long terminal repeat (LTR). The exposed X-ray film revealed about 370 distinct spots in the 2-D maps. In comparing the 2-D maps, genome rearrangement involving IAP was detected. o 1990 Academic
with
Contract
DE-
the mid-repetitive sequences ( lo’-lo3 copies/genome in mammalian cells). This approach is based on the ability to separate the restriction fragments of genomic DNA in the agarose gel by a Z-D3 pattern (10). After Southern blotting, the DNA on the filter was hybridized with a labeled end fragment of a mid-repetitive sequence. Comparing the 2-D restriction fragment maps of one test cell type versus that of another reference cell type, we hope to be able to monitor any rearrangement around the mid-repetitive sequences between these two cell types in question. This method will be useful in investigating the role of DNA rearrangement in differentiation and/or carcinogenesis involving the mid-repetitive sequences. For this study we have monitored the intracisternal A particle (IAP) gene. IAP genes are endogenous retrovirus-like sequences found in rodents (11,12), with 102lo3 copies per genome. IAP genes are dispersed among all the chromosomes (13). These sequences are structurally similar to the transposons having a long terminal repeat (LTR) at each end. Some of the LTRs have transcriptional promoter and enhancer activities (14,X). Endogenous viral sequences do activate cellular genes including protooncogenes in some cases (16-B). On the basis of the reasons mentioned above, monitoring the genomic DNA rearrangement involving IAP genes would be of value. MATERIAL
AND
METHODS
Cell Cultures The cell line PC44 was provided by Dr. Yasushi Yokokawa of our division. This cell line was derived from a clone of an established diploid myoblast cell line pre3 Abbreviations used: IAP, intracisternal A particle; Z-D, mensional; BSA, bovine serum albumin; LTR, long terminal SDS, sodium dodecyl sulfate; BSA, bovine serum albumin.
two direpeat;
0003-2697/90
$3.00
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
MID-REPETITIVE
SEQUENCES
AS PROBES
3’L 2
TR
FIG. 1. The probe and its location in the map of an IAP gene from Syrian hamster. Abbreviations: B, BglII; H, HindIII; P, PstI.
pared from g-day gestation Syrian hamster embryo cells by continuous treatment with phorbol 12,13-didecanoate at 0.1 pg/ml(l9). The PC44 cells were cultivated in ERM (Dulbecco’s Eagle reinforced medium) with 20% fetal calf serum. Another cell line, BPGT was derived from the same isogenic Syrian hamster as PC44. BPGT is a highly tumorigenic, chemically transformed clone of Syrian hamster embryo cells (20). Extraction
of High Molecular
Weight DNA
Cells from two to four T150 flasks were trypsinized and collected by centrifugation. The cells were lysed in 1 ml of cell lysis buffer (20 mM Tris, pH 8, 0.1 M NaCl, 0.5% v/v Nonidet P-40, 10% sucrose). The cell lysate was transferred to a l&ml centrifuge tube and was overlayered on 2 ml of cell lysis buffer containing 30% sucrose. Nuclei were pelleted by centrifugation (10K rpm, 10 min, in HB-4 rotor). The nuclei were lysed in 2 ml of proteinase K buffer (0.5% SDS, 10 mM EDTA, 50 mM Tris, pH 8) containing 2 mg of proteinase K. The hydrolysis was carried out at 37°C for 2.5 h. One-tenth volume of 5 M NaClO, was then added and lysate was extracted once with phenol:chloroform (1:l) and then twice with chloroform. The DNA solution was dialyzed against 1X SSC overnight. After dialysis, the DNA solution was treated with RNase A (50 pg/ml) at 37°C for 1 h. One-third volume of 4~ proteinase K buffer was added, followed by proteinase K digestion (100 pg/ml) at 37°C for 1 h. The DNA solution was extracted once with phenol:chloroform (1:l) and twice with chloroform. After the addition of l/9 vol of 3 M sodium acetate, pH 7,2 vol of 95% alcohol (-20°C) was overlayered on the DNA solution. DNA was spooled out by a glass rod. DNA was then redissolved by dialyzing against TE buffer (5 mM Tris-HCl, 1 mM EDTA, pH 8.2).
IN
GENOME
REARRANGEMENT
327
with EcoRI reaction buffer (0.1 M Tris, pH 7.7, 50 mM NaCl, 10 mM MgCl,) plus 250 pg/ml bovine serum albumin (BSA) by shaking 1 h in room temperature. Then the gel strip was put in a small column (total volume, I5 ml) which contained EcoRI reaction buffer plus 250 pg/ ml BSA and 200 units/ml EcoRI. The column was gently rotated for 20 h at 37°C. After the EcoRI in situ digestion, the gel strip was equilibrated with IX TPE buffer. The gel strip was then fused with a 1% gel slab which has been equilibrated with 3X TPE, 25% formamide, by adding melted agarose (1% in TPE) to fill up the gap. Electrophoresis was carried out at 4°C in 1X TPE buffer. After electrophoresis, the gel was stained with ethidium bromide. Southern
Blot and Probe Hybridization
The Southern blot, hybridization, and washing protocols were carried out in accordance with the previously described methods (21). The DNA fragments in the 2-D gel were transferred onto Gene Screen membrane by Southern blotting. After baking, the membrane was prehybridized in hybridization buffer (0.45 M NaCl/ 0.045 M sodium citrate/O.l% SDS/0.02% polyvinylpyrrolidone/0.02% Ficoll/O.O2M potassium phosphate, pH 6.8) containing 100 @g/ml of sheared and denatured herring sperm DNA at 61°C for 5 h. The hybridization buffer containing 1 Fg of 32P-labeled IAP probe (see below) at 61°C for 18 h. The washing procedure was carried out at 61°C. Probe The 1.9-kb IAP Hind111 fragment of Syrian hamster (Fig. 1) was provided by Dr. Anne Brown (unpublished data). The 1.9-kb IAP fragment contains the same BglII and P&I restriction sites and orders as described by Ono and Toh (22). The 350-bp BglIIIPstI fragment was isolated and labeled by nick translation. The reaction mixture contains 1 pg probe DNA, 50 I’IIM NaPi, pH 6, 10 mM MgCl,, 1 mM EDTA, 60 PM dATP and dGTP, and 50 &i [a-32P]dCTP (3000 Ci/mmol), and 25 units of DNA polymerase I (Boehringer-Mannheim Biochemicals). The mixture was incubated at room temperature for 6 min and then shifted to 13’C for 2 h. The specific activity was 3.5 X lo7 dpm/pg DNA.
2-D Agarose Gel of Genomic Restriction Fragments
RESULTS
High molecular weight DNA was digested completely by BgZII. The digested DNA was loaded in a well of a 1% agarose gel (length, 25 cm; width, 20 cm; thickness, 0.46 cm). Electrophoresis was carried out in 1X TPE buffer (0.08 M Tris-phosphate, 0.008 M EDTA, pH 7.7). After first-dimension electrophoresis, the DNA fragments in the agarose gel were digested with EcoRI by the following method: the gel strip was first equilibrated
Encouraged by the results of 2-D mapping of restriction fragments of Escherichia coli genomic DNA (lo), we tried to extend our work to the mammalian cell for creating a 2-D map of mid-repetitive sequence. High molecular weight genomic DNA from Syrian hamster was digested completely by BgZII. An ll-pg sample of digested DNA was loaded in a well of 1% agarose gel. The procedure for generating a 2-D gel map of genomic
AND
DISCUSSION
328
AU,
23 I
94 I
66 I
TS’O,
44 I
AND
YI
20 I 23
94
23 I
94 I
66 I
44 I
20 I 23
94
20 FIG. 2.
The Z-D restriction fragments maps of the IAP 5’ end containing sequences of PC44 (a), and BPGT (c). The procedure is described under Materials and Methods. The small arrows point out the difference in spots between the two maps. The arrow pointed upwards indicates the presence of a spot and that pointed downwards indicates the absence of a spot. Control for the full digestion, 1 pg of X phage DNA was electrophoresed into the gel. The piece of gel containing the DNA was cut and underwent EcoRI in-gel digestion together with the experimental gel strip. The digested fragments were revealed after electrophoresis (b).
FIG. 3. experiments.
Two
2-D restriction fragment maps of the genomic DNA PC44 cells The patterns of various clusters of spots can be clearly recognized.
with
the
IAP
5’ end
as probes
showing
reproducibility
of two
330
AU,
TS’O,
restriction fragments was followed (see Materials and Methods). After ethidium bromide staining, only a few spots were resolved (data not shown), as compared with the 2-D gel maps of E. coli genomic DNA (10). This lack of resolution was due to crowding of too many spots overlapping with each other. Only the highly repetitive sequences can be seen as distinct spots as revealed by staining. Subsequently the Syrian hamster genomic DNA fragments in the 2-D gel were transferred to membrane by Southern blotting and hybridized with IAP probe as described under Materials and Methods. The probe used was a 350-bp BgZII/PstI fragment cloned from a Syrian hamster IAP gene (Fig. 1). This probe is close to but does not overlap with the 5’ LTR, and thus, this probe can only detect the sequences flanking the 5’ LTR but cannot detect the sequences flanking the 3’ LTR. In view of the diversity of IAP genes in the genome, we selected less stringent conditions (61’C) for the probe hybridization. About 370 distinct spots were revealed after autoradiography (Fig. 2A). The difference in intensities of these spots observed in the 2-D map may be caused by differences in the extent of homogeneity between these individual IAP sequences and the probe used in this experiment. In addition, some IAP sequences may be reiterated in tandem in the genome. Sequences with a higher copy numbers will bring about higher intensity. Since the DNA was not evenly distributed in the gel, a control was carried out for every in-gel digestion in order to ensure that all the DNA fragments in the gel strip were fully digested. A l-bg sample of X phage DNA was electrophoresed into the gel. The piece of gel containing the X phage DNA was cut and underwent in-gel digestion together with the gel strip containing the hamster DNA. Since the X DNA concentration at the band is higher than hamster DNA at any place in the gel strip, the full digestion of that X band, as shown by electrophoresis (Fig. 2B), ensures the full digestion of DNA in the gel strip. The dark strip appearing at diagonal place represents the IAP-containing DNA restriction fragments which did not have EcoRI cutting site. A rearranged IAP-containing fragment might not be detected if this fragment does not have the EcoRI cutting site and thus locates at the dark strip. In this case, different restriction enzymes (for the first or second digestion) should be considered. This procedure has been shown to be highly reproducible. The PC44 genomic DNA have been analyzed four times through this 2-D gel electrophoresis procedure. The results have been remarkably similar and two of the four gel patterns are shown in Fig. 3. The genomic DNA for BPGT were also analyzed repeatedly in yielding very similar patterns. Polymorphism in DNA sequence exists in individuals of some species, so the DNA samples for the 2-D com-
AND
YI
parison should be obtained from the same animal or from isogenic animals. The question of changes in DNA methylation should also be considered. The restriction enzymes chosen for this experiment, EcoRl and BglII, are not sensitive to the methylation of -GGCCor GCGC- sequences. On the other hand, by the choice of appropriate enzymes pairs, the change of DNA methylation pattern can be the subject of the investigation instead of DNA rearrangement. In summary, a highly reproducible procedure is demonstrated here for monitoring mammalian genome rearrangement. This procedure utilizes two restriction enzyme treatments and 2-D gel electrophoresis for separation and uses the appropriate portion (ends) of a midrepetitive DNA sequence as probes in Southern blotting for detection. This procedure has the capability of monitoring up to 1000 regions of the genome for rearrangement adjacent to a chosen repetitive DNA sequence which are hotspots for such activities. REFERENCES 1. Flavell,
I.-H.
2. Doring, 127-130.
H. P., and
3. Baltimore,
(1983)
Cell 34,415-419. Tillmann,
D. (1985)
E. (1984)
Nature
(London)
30’7,
Cell 40,481-482.
4. Fasullo, M. T., and Davis, R. W. (1987) Proc. N&l. Acud. Sci. USA 84,6215-6219. 5. Kupiec, M., and Petes, T. D. (1988) Genetics 119,549-559. 6. Stark, G. R., Debatisse, Cell 5’7, 901-908.
M.,
Giulotto,
E., and Wahl,
G. M. (1989)
I. Latt, S. (1981) Annu. Reu. Genet. 15, l-57. 8. Lerman, M. A., Schneider, W. J., Sudhof, T. C., Brown, M. Coldstein, J. L., and Russell, D. W. (1985) Science 227,140-146. 9. Uitterlinden, (1989) Proc.
A. G., Slagboom, P. E., Knook, Natl. Acad. Sci. USA 86,2742-2746.
10. Yi, M., Au, L., Ichikawa, Sci. USA 87,3919-3923. 11. Calarco, 93.
N., and Ts’o,
P. G., and Szollosi,
P. (1973)
P. (1990) Nature
S.,
D. L., and Vijg, Proc. Natl. (London)
12. Chase, P. G., and Piko, L. (1973) J. N&l. Cancer Inst. 1973. 13. Lueders, K. K., and Kuff, E. L. (1977) Cell 12,965-972.
J.
Acad.
243,91-
51, 1971-
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