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Analysis of protein binding in the MLL translocation breakpoint region.

Objective: The MLL gene is remarkable for its involvement in translocations with greater than 50 unique loci in several different subtypes of human acute leukemia. In addition, the region of MLL that breaks and joins to other genes in leukemogenic leu·ke·mo·gen·ic
adj.
1. Of or relating to leukemogenesis.

2. Of, relating to, or characterized by a leukemogen.


leukemogenic adjective
 translocations is remarkably conserved at just over 8 kilobases (kb) in a central region of the gene. This 8 kb region is referred to as the MLL breakpoint The location in a program used to temporarily halt the program for testing and debugging. Lines of code in a source program are marked for breakpoints. When those instructions are about to be executed, the program stops, allowing the programmer to examine the status of the program  cluster region (bcr).

Much sequence and structure analysis has been done to characterize the MLL bcr to elucidate the mechanism of MLL translocation. Various studies have shown evidence for potential recombination mediating features, including a DNA topoisomerase II cleavage site, a DNase I hypersensitivity site and recombination signal sequences for V(D)J gene segment joining. In addition, the region is rich in repetitive DNA DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 with several Alu repeats which could facilitate homologous recombination with loci throughout the genome.

However, little is known about the protein-binding characteristics of this region. We have begun analyzing protein binding in the MLL bcr using electrophoretic mobility shift assay An electrophoretic mobility shift assay (EMSA), also referred as a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common technique used to study protein-DNA or protein-RNA interactions.  (EMSA). Detection of differential protein binding potential within the bcr may provide insight into the regions of the MLL bcr which are necessary for recombination with other loci. More important, eventual identification of specific MLL bcr binding proteins would provide essential clues to the mechanism of aberrant recombination involving MLL.

Methods: Nuclear extracts from hematopoietic hematopoietic /he·ma·to·poi·et·ic/ (-poi-et´ik)
1. pertaining to hematopoiesis.

2. an agent that promotes hematopoiesis.


hematopoietic

1. pertaining to or affecting the formation of blood cells.
 and non-hematopoietic cells are incubated with labeled double stranded oligonucleotides from 50-200 bp in length in EMSA binding buffer. Binding of proteins to specific MLL bcr oligonucleotides is indicated by retarded migration in 6% polyacrylamide gels relative to oligonucleotide migration in the absence of proteins. Specific binding is shown by competition with unlabeled oligonucleotides.

Results: Binding of nuclear protein(s) has been detected in the extreme 5' end of the 8 kb MLL bcr. The binding is sequence specific as shown by competition with unlabeled oligonucleotide of the same sequence. Binding was seen with nuclear extracts from two hematopoietic cell lines, REH and Jurkat, but not with a purified control protein (OCT2 transcription factor). The degree of retardation of migration indicates a possible protein complex binding the 5' oligonucleotide. Other regions of the MLL bcr, including the extreme 3' boundary of the MLL bcr and a region described as an in vivo DNA topoisomerase II cleavage site were did not show retarded migration in EMSA with REH or Jurkat nuclear extracts.

Conclusions: We have identified one apparent region of protein binding

within the MLL bcr. The region marks the extreme 5' boundary of the bcr and may be relevant in the formation of MLL translocations. Experiments are underway to narrow the 200-bp region of binding by using small overlapping oligonucleotides from the region and to test binding with nuclear extracts from non-hematopoietic cell lines. Ultimately protein binding will be compared throughout the MLL bcr with the final goal of identifying potential recombination-mediating nuclear proteins.

Heidi J. Super, Alysa L. Anderson and Aileen Aldrich

Department of Biology, Minot State University Minot State University (MSU or MiSU) is a four-year institution of higher learning in Minot, North Dakota. Founded in 1913 as a normal school, Minot State University is the third-largest public university in North Dakota, offering undergraduate and graduate degree , Minot ND 58707
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Title Annotation:COMMUNICATIONS--PROFESSIONAL
Author:Super, Heidi J.; Anderson, Alysa L.; Aldrich, Aileen
Publication:Proceedings of the North Dakota Academy of Science
Date:Apr 1, 2007
Words:500
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