Restriction Enzyme Analysis of DNA Methylation in “Condensed” Chromatin of Ha-Ras-Transformed NIH 3T3 Cells
Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha‐ras oncogene. The structural basis for this oncogene‐mediated alteration in nuclear organization is unknown. S...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2000-01-01
|
| Series: | Analytical Cellular Pathology |
| Online Access: | http://dx.doi.org/10.1155/2000/138230 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832554459971977216 |
|---|---|
| author | Maria Luiza S. Mello Ann F. Chambers Benedicto C. Vidal Wolfgang Planding Ulrich Schenck |
| author_facet | Maria Luiza S. Mello Ann F. Chambers Benedicto C. Vidal Wolfgang Planding Ulrich Schenck |
| author_sort | Maria Luiza S. Mello |
| collection | DOAJ |
| description | Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha‐ras oncogene. The structural basis for this oncogene‐mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher‐order chromatin organization induced by Ha‐ras. CpG‐methylated DNA content was estimated in “condensed” chromatin of Ha‐ras‐transformed NIH 3T3 cell lines which differ in ras expression and ras‐induced metastatic ability but present approximately the same values of “condensed” chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher‐order organization induced by Ha‐ras in these cell lines, the methylated DNA density in the “condensed” chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen‐stained cells previously subjected to treatment with Msp I and Hpa II restriction enzymes, which distinguish between methylated and non‐methylated DNA. The amount of methylated CpG sequences not digested by Hpa II in “condensed” chromatin regions was found to vary in the studied ras‐transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced by ras transformation in the mentioned NIH 3T3 cell lines. |
| format | Article |
| id | doaj-art-d3c09e6893f04e758626f5753c169d54 |
| institution | Kabale University |
| issn | 0921-8912 1878-3651 |
| language | English |
| publishDate | 2000-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Analytical Cellular Pathology |
| spelling | doaj-art-d3c09e6893f04e758626f5753c169d542025-02-03T05:51:28ZengWileyAnalytical Cellular Pathology0921-89121878-36512000-01-0120416317110.1155/2000/138230Restriction Enzyme Analysis of DNA Methylation in “Condensed” Chromatin of Ha-Ras-Transformed NIH 3T3 CellsMaria Luiza S. Mello0Ann F. Chambers1Benedicto C. Vidal2Wolfgang Planding3Ulrich Schenck4Department of Cell Biology, Institute of Biology, UNICAMP, 13083‐970 Campinas, SP, BrazilDivision of Experimental Oncology, Department of Oncology, University of Western Ontario and London Regional Cancer Centre, London, Ontario, N6A 4L6, CanadaDepartment of Cell Biology, Institute of Biology, UNICAMP, 13083‐970 Campinas, SP, BrazilLaboratory of Clinical Cytology, Institute of Pathology, Technical University of Munich, 81675 Munich, GermanyLaboratory of Clinical Cytology, Institute of Pathology, Technical University of Munich, 81675 Munich, GermanyIncreased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha‐ras oncogene. The structural basis for this oncogene‐mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher‐order chromatin organization induced by Ha‐ras. CpG‐methylated DNA content was estimated in “condensed” chromatin of Ha‐ras‐transformed NIH 3T3 cell lines which differ in ras expression and ras‐induced metastatic ability but present approximately the same values of “condensed” chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher‐order organization induced by Ha‐ras in these cell lines, the methylated DNA density in the “condensed” chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen‐stained cells previously subjected to treatment with Msp I and Hpa II restriction enzymes, which distinguish between methylated and non‐methylated DNA. The amount of methylated CpG sequences not digested by Hpa II in “condensed” chromatin regions was found to vary in the studied ras‐transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced by ras transformation in the mentioned NIH 3T3 cell lines.http://dx.doi.org/10.1155/2000/138230 |
| spellingShingle | Maria Luiza S. Mello Ann F. Chambers Benedicto C. Vidal Wolfgang Planding Ulrich Schenck Restriction Enzyme Analysis of DNA Methylation in “Condensed” Chromatin of Ha-Ras-Transformed NIH 3T3 Cells Analytical Cellular Pathology |
| title | Restriction Enzyme Analysis of DNA Methylation in “Condensed” Chromatin of Ha-Ras-Transformed NIH 3T3 Cells |
| title_full | Restriction Enzyme Analysis of DNA Methylation in “Condensed” Chromatin of Ha-Ras-Transformed NIH 3T3 Cells |
| title_fullStr | Restriction Enzyme Analysis of DNA Methylation in “Condensed” Chromatin of Ha-Ras-Transformed NIH 3T3 Cells |
| title_full_unstemmed | Restriction Enzyme Analysis of DNA Methylation in “Condensed” Chromatin of Ha-Ras-Transformed NIH 3T3 Cells |
| title_short | Restriction Enzyme Analysis of DNA Methylation in “Condensed” Chromatin of Ha-Ras-Transformed NIH 3T3 Cells |
| title_sort | restriction enzyme analysis of dna methylation in condensed chromatin of ha ras transformed nih 3t3 cells |
| url | http://dx.doi.org/10.1155/2000/138230 |
| work_keys_str_mv | AT marialuizasmello restrictionenzymeanalysisofdnamethylationincondensedchromatinofharastransformednih3t3cells AT annfchambers restrictionenzymeanalysisofdnamethylationincondensedchromatinofharastransformednih3t3cells AT benedictocvidal restrictionenzymeanalysisofdnamethylationincondensedchromatinofharastransformednih3t3cells AT wolfgangplanding restrictionenzymeanalysisofdnamethylationincondensedchromatinofharastransformednih3t3cells AT ulrichschenck restrictionenzymeanalysisofdnamethylationincondensedchromatinofharastransformednih3t3cells |