Figure 3.
Figure 3. Reduced NEMO expression was caused by duplication of the NEMO gene. (A) Southern blotting analysis of the NEMO gene. Genomic DNA was digested with HindIII and electrophoresed, and the blot was hybridized with exon 2 DNA as a probe. DNA from the patient's EBV-transformed cells (lane 3) showed a 15-kb band, which was shared by his mother (lane 2), instead of the 11-kb bands, which were detected in the DNA from the patient's HTLV-I-transformed cells (lane 4), his father's EBV-transformed cells (lane 1), his mother's EBV-transformed cells (lane 2), and EBV-transformed cells from healthy controls (lanes 5-7). (B) Northern blotting analysis of the NEMO gene. The blot was hybridized with exon 3 DNA as a probe. RNA from the patient's EBV-transformed cells (lane 2) expressed very little NEMO mRNA compared with his HTLV-I-transformed cells (lane 1) and the EBV-transformed cells from healthy controls (lanes 3-4). (C) PCR reaction using primers 1 and 7. Only the NEMO gene was amplified. EBV-transformed cells (lane 2) had a 13-kb band instead of the 9-kb band seen in HTLV-I-transformed cells (lane 1) and healthy controls (lanes 3-4). M indicates lambda/HindIII digestion marker. (D) PCR reaction using primers 1 and 2 (lanes 1-2), primers 1 and 3 (lanes 3-4), primers 1 and 4 (lanes 5-6), primers 1 and 5 (lanes 7-8), primers 1 and 6 (lanes 9-10), and primers 1 and 7 (lanes 11-12). The patient's EBV-transformed cells (lanes 2, 4, 6, 8, 10, and 12) and those of healthy controls (lanes 1, 3, 5, 7, 9, 11) were used as templates. The larger bands detected in lanes 8, 10, and 12 indicate that the insertion is between primers 4 and 5 (between introns 5 and 6). M indicates 2.5-kb ladder. (E) PCR reaction using primers 8 and 5 (lanes 1-2), primers 8 and 3 (lanes 3-4), and primers 8 and 2 (lanes 5-6). The patient's EBV-transformed cells (lanes 2, 4, and 6) and those of healthy controls (lanes 1, 3, and 5) were used as templates. M indicates 1-kb marker. (F) Schematic representation of the normal NEMO gene, the patient's NEMO gene, and the NEMO pseudogene. Primers are shown as triangles. The duplicated region found in the patient is shaded. H indicates HindIII digestion sites.

Reduced NEMO expression was caused by duplication of the NEMO gene. (A) Southern blotting analysis of the NEMO gene. Genomic DNA was digested with HindIII and electrophoresed, and the blot was hybridized with exon 2 DNA as a probe. DNA from the patient's EBV-transformed cells (lane 3) showed a 15-kb band, which was shared by his mother (lane 2), instead of the 11-kb bands, which were detected in the DNA from the patient's HTLV-I-transformed cells (lane 4), his father's EBV-transformed cells (lane 1), his mother's EBV-transformed cells (lane 2), and EBV-transformed cells from healthy controls (lanes 5-7). (B) Northern blotting analysis of the NEMO gene. The blot was hybridized with exon 3 DNA as a probe. RNA from the patient's EBV-transformed cells (lane 2) expressed very little NEMO mRNA compared with his HTLV-I-transformed cells (lane 1) and the EBV-transformed cells from healthy controls (lanes 3-4). (C) PCR reaction using primers 1 and 7. Only the NEMO gene was amplified. EBV-transformed cells (lane 2) had a 13-kb band instead of the 9-kb band seen in HTLV-I-transformed cells (lane 1) and healthy controls (lanes 3-4). M indicates lambda/HindIII digestion marker. (D) PCR reaction using primers 1 and 2 (lanes 1-2), primers 1 and 3 (lanes 3-4), primers 1 and 4 (lanes 5-6), primers 1 and 5 (lanes 7-8), primers 1 and 6 (lanes 9-10), and primers 1 and 7 (lanes 11-12). The patient's EBV-transformed cells (lanes 2, 4, 6, 8, 10, and 12) and those of healthy controls (lanes 1, 3, 5, 7, 9, 11) were used as templates. The larger bands detected in lanes 8, 10, and 12 indicate that the insertion is between primers 4 and 5 (between introns 5 and 6). M indicates 2.5-kb ladder. (E) PCR reaction using primers 8 and 5 (lanes 1-2), primers 8 and 3 (lanes 3-4), and primers 8 and 2 (lanes 5-6). The patient's EBV-transformed cells (lanes 2, 4, and 6) and those of healthy controls (lanes 1, 3, and 5) were used as templates. M indicates 1-kb marker. (F) Schematic representation of the normal NEMO gene, the patient's NEMO gene, and the NEMO pseudogene. Primers are shown as triangles. The duplicated region found in the patient is shaded. H indicates HindIII digestion sites.

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