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First published online August 26, 2004
doi: 10.1242/10.1242/jcs.01332

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Breakpoint cluster regions of the AML-1 and ETO genes contain MAR elements and are preferentially associated with the nuclear matrix in proliferating HEL cells

Laboratory of Structural and Functional Organization of Chromosomes, Institute of Gene Biology RAS, Vavilov Street 34/5, 119334, Moscow, Russia

View larger version (96K): [in a new window]   Fig. 1. Fluorescent in situ hybridization of AML-BCR3 (A,A') and ETO-BCR2 (B,B') probes with nuclear halos immobilized on microscopic slides. (A',B') The nuclei were pretreated with RNase A before high-salt extraction. Hybridization signals are presented as black spots (arrows) superimposed on the nuclear halos stained with DAPI.

View larger version (78K): [in a new window]   Fig. 2. (A-A'') Identification of the nuclear matrix borders by immunostaining of lamins on nuclear halos immobilized on microscopic slides. (A) Nuclear halo stained with DAPI. (A') Nuclear halo immunostained using antibodies against lamins A and B. (A'') Superimposition of (A,A'). (B-B'') Hybridization in situ of Alu repeat to a nuclear halo. (B) Nuclear halo stained with DAPI. (B') Results of hybridization (immunostaining of biotinylated probe). (B'') Superimposition of (A,A').

View larger version (53K): [in a new window]   Fig. 3. Analysis of the transcriptional status of ETO and AML-1 genes in HEL cells using RT-PCR. Each pair of primers was first tested on total DNA to provide a positive control (`DNA') and then used to amplify the test fragments on the template synthesized by reverse transcription (starting from non-specific primers) of total RNA from HEL cells (`RNA RT+'). The lanes designated `RNA RT–' represent a negative control loaded with amplification products synthesized without a cDNA template. All reactions were set up as in the previous case, but the reverse transcription enzyme was omitted from the first-strand synthesis mixture. Molecular sizes of the marker bands are shown at the right side of the lane loaded with the marker (M).

View larger version (92K): [in a new window]   Fig. 4. Fluorescent in situ hybridization of AML-control (A) and ETO-control (B) probes with nuclear halos immobilized on microscopic slides. Hybridization signals are presented as black spots (arrows) superimposed on the nuclear halos stained with DAPI.

View larger version (22K): [in a new window]   Fig. 5. Amplification of different AML-1 gene regions using total DNA and nuclear matrix DNA as templates. At the top is a map of the gene showing the positions of three BCRs and four test regions (vertical bars). The results of PCR amplifications of each test region on the total DNA template (T) and the nuclear matrix DNA template (M) are presented below the map. The figures below the images of the amplified fragments show the amount of DNA in the test fragments amplified on the nuclear matrix DNA template relative to the amount of DNA in the test fragments amplified on the total DNA template. The figures represent the average of the results obtained in three independent experiments.

View larger version (35K): [in a new window]   Fig. 6. Mapping of MAR elements in AML-BCR3 and ETO-BCR2. Different fragments present in the input mixture are shown by arrows at the left-hand side of the lane with input DNA. The other four lanes in each experiment represent the fragments obtained from the nuclear matrix incubated with the input fragments in the presence of increasing amounts (50 µg ml–1, 100 µg ml–1, 200 µg ml–1 and 500 µg ml–1) of cold, non-specific competitor DNA (sheared Escherichia coli DNA), whereas the amount of labeled input DNA was constant (1 µg ml–1) in all cases. Notice that the 900 bp subfragment of ETO-BCR2 and the 1200 bp subfragment of AML-BCR3 are retained by nuclear matrices to the same extent as bona fide MARs from the Drosophila histone gene cluster (`Hist-MAR'), whereas the other subfragments of the BCRs and the pUC DNA are washed out at high concentrations of non-specific competitor DNA.

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