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Molecular analysis of the MLL breakpoint region in leukemia: a study of binding of DNA-topoisomerase II and/or other nuclear proteins to specific DNA breakpoint fragments.

A common phenomenon of leukemia cells is specific, non-random, reciprocal chromosome translocations, in which two non-homologous chromosomes exchange pieces of DNA with specific breakpoints in each chromosome. As a result of the translocation, the regulation of the gene at the breakpoint is either altered, creating too much or too little of the gene product, or a whole new fusion gene can be created, producing a new fusion gene product, with altered activity. Either of these scenarios can set off malignant transformation of cells and contribute to the development of leukemia. Many chromosomal translocations correlate with a specific subtype of leukemia. The MLL gene is located at the chromosome breakpoint in translocations on the long arm of chromosome 11 (band q23). The MLL gene is involved in translocations with 45 other chromosomal regions including specific identified genes on chromosomes 4, 6, 9, 19, X. In all studies so far the 5' end of the MLL gene becomes fused to the 3' end of another gene creating an oncogenic MLL fusion gene and protein. The MLL translocation breakpoint is 8000 base pairs in length and contains exons 5-11 of the MLL gene. MLL translocations occur in both acute childhood leukemias and in adult leukemias that arise following treatment of a primary tumor. Therapy-related leukemias develop following treatment with a cellular enzyme DNA-topoisomerase II (topo II) inhibiting drug.

The restricted size of the MLL breakpoint region, only 8kb, suggests that the mechanism of the translocations involve a specific DNA sequence(s) in the region. Several studies have suggested that the MLL breakpoint region is prone to cleavage by topo II, but the region has not yet been analyzed for direct binding of topo II or other nuclear proteins. We have used a standard gel shift mobility assay to determine if topo II protein and/or nuclear proteins normally bind to the MLL breakpoint region. Using Polymerase Chain Reaction (PCR) we amplified three 200 base pair DNA fragments from the breakpoint region. Two of the fragments, probes A and C, lie at the extreme 5' and 3' ends of the breakpoint region, respectively. Our third probe, B, is located at a proposed cleavage site for topo II in the MLL gene. Probes A, B, and C were labeled for chemiluminescent detection and then incubated with nuclear proteins from a human leukemia cell line, REH, in separate experiments. The binding of nuclear proteins to the probes was determined by retarded migration (shifting) of probes during polyacrylamide gel electrophoresis.

Our analysis indicates that probe A specifically binds one or more nuclear proteins from the REH cell line. In a modified "supershift" assay we tested the binding of the topo II antibody by adding a polyclonal antibody to topo II to the reaction. We preliminarily noted additional shifting of the probe, suggesting that the protein topo II is binding to probe A. Since topo II antibody binding was inconclusive and suitable antibody controls were not available, we took a more direct approach of using the purified topo II protein in the electrophoretic mobility shift assay. No shifting was detected using probe A and purified topo II, indicating a different nuclear protein is binding this region of the MLL breakpoint. From previous studies we had predicted that probe B would possibly bind topo II directly, but after many trials we did not detect binding of REH nuclear proteins to either probes B or C.

The correlation of topo II binding and MLL translocations is still a possibility. There are many more regions of the 8kb MLL breakpoint region yet to be analyzed for binding of topo II and/or nuclear proteins. Future studies will also focus on identification of the nuclear protein binding probe A.

Jessica D. Hamilton * and Heidi J. Super

Department of Biology, Minot State University, Minot, ND 58707
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Title Annotation:A. Rodger Denison Student Research Competition: COMMUNICATIONS: UNDERGRADUATE DIVISION
Author:Hamilton, Jessica D.; Super, Heidi J.
Publication:Proceedings of the North Dakota Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2004
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