Figure 2.

(A) Results of RT-PCR analyses. Lane M contains a size marker VI (Boehringer Mannheim). Lanes 1, 4, 7, and 12 are negative controls in which the cDNA was replaced by sterile water. Normal fragments are obtained from the patient's unaffected GRAF allele (lane 2) and from the cell line Mono-Mac6 (lane 3). A MLL/GRAF fusion mRNA is detected in the sample with a t(5;11)(q31;q23) (lane 5) but not in the cell line lacking this translocation (lane 6). Normal fragments are obtained from the patient's unaffected MLL allele (lane 8) and the cell line Mono-Mac6 (lane 9). The additional fragments in lanes 5, 8, and 9 are generated by MLL splice variants. Further analysis reveals that the reciprocal GRAF/MLL fragment is neither present in the patient's sample (lane 10) nor in the cell line (lane 11). Control amplifications with primers specific for the ABL gene are shown in lanes 15 (patient sample) and 16 (cell line). (B) Long-range PCR results of genomic DNA. Lanes M contain the size markers III and VI (Boehringer Mannheim). Lane 1 is a negative control. A MLL/GRAF fusion product is detected in the patient with the t(5;11)(q31;23) (lane 2) but not in the control cell line Mono-Mac6 (lane 3). Lanes 4, 5, 9, and 17 are negative controls. A normal 8-kb fragment that covers the breakpoint cluster region of the unaffected MLL alleles in the patient with the t(5;11)(q31;q23) (lane 6), in a healthy individual (lane 7), and in the Mono-Mac cell line (lane 8) is seen. No reciprocal GRAF/MLL gene fragment is detected in any of these samples (lanes 10–13), whereas in all of them an approximately 13-kb long intron of GRAF becomes evident (lanes 14–16). (C) Sequence and schematic representation of the inverted duplication of MLL within the genomic MLL/GRAF fusion. Numbering of nucleotides within the breakpoint region of MLL according to ref. 23. The horizontal arrows indicate the positions of the primers used for amplification of the genomic MLL/GRAF fusion seen in lane 2 of B.