PMC

Localization of GFP-BLM fusion proteins. The immunofluorescent images show representative cells from each transfected cell line. Localization of GFP or GFP-BLM fusion proteins and PML (α-PML and Donkey α-mouse Texas Red secondary) or TOPIIIα (α-TOPIIIα and Donkey α-rabbit Texas Red secondary) in DAPI-stained nuclei are shown. A. EGFP + α-PML. B. GFP-BLM + α-PML. C. GFP-BLM + α-TOPIII. D. GFP-ΔN1 + α-PML. E. GFP-ΔN2 + α-PML. F. GFP-ΔN2 + α-TOPIII. G. GFP-ΔN3 + α-PML. H. GFP-ΔN4 + α-PML. L GFP-K695T + α-PML. J.GFP-ΔH1 + α-PML K. GFP-ΔC1 + α-PML. L. GFP-ΔC2 + α-TOPIII. The parental SV40-transformed BS cell line lacks detectable BLM [].

SCE Assay on cell lines with and without inducer.
A. Histogram of the distribution of SCEs from induced cell lines. The number of SCEs per 46 chromosomes is plotted vs. the number of cells or metaphases. B. Graphical representation of the relative correction for each GFP-BLM allele (Table &). C. Metaphase chromosome spread from an induced cell transfected with GFP-ΔC1 BLM. A large ring chromosome is shown at the top of the spread. The SCEs are visualized with acridine orange staining.

BLM Mutation Map. A diagram of the of the deletion and missense alleles of BLM used in this study. The amino acids removed by each deletion are: ΔN1 (2-128), ΔN2 (135-235), ΔN3 (241-469), ΔN4 (402-600), ΔH1 (903-1115), ΔC1 (1118-1164) and ΔC2 (1166-1331). The location of the missense allele K695T is shown. The red boxes indicate positions of regions of the N-terminus of BLM that have a high density of aspartic and glutamatic acid residues: region 1 (261-310 = 42% DE) and region 2 (541-570 = 50% DE). Deletion N3 removes a serine-rich region (310-320 = 64%S). The blue box indicates the location of the nuclear localization signal [] (NLS = 1331-1350). The two regions of reported in vitro interaction of BLM with TopIIIα (143-213 & 1266-1417) are marked []. The helicase domain consists of residues 672-985 and the HRD (Helicase and RNaseD identity domain) [] is indicated (1212-1293).

Western analysis of stable transfected cell lines expressing mutant BLM alleles. A. Whole cell lysates (15 μg total protein) from uninduced and induced cells were resolved on 4-12% SDS polyacrylamide gels and transferred to PVDF membranes. The bound complexes were detected with ECL reagents and exposed to x-ray film: 1) GFP-BLM+Tet, 2) GFP-ΔN1 no Tet, 3) GFP-ΔN1+Tet, 4) GFP-ΔN2 no Tet, 5) GFP-ΔN2+Tet. 6) GFP-ΔN3 no Tet, 7) GFP-ΔN3+Tet, 8) GFP-ΔN4 no Tet, 9) GFP-ΔN4+Tet. B. ECL western analysis of whole cell lysates: 1) GFP-BLM+Tet, 2) GFP-K695T no Tet, 3) GFP-K695T+Tet, 4) GFP-ΔH1 no Tet, 5) GFP-ΔH1+Tet, 6) GFP-ΔC1 no Tet, 7) GFP-ΔC1+Tet, 8) GFP-ΔC2 no Tet, 9) GFP-ΔC2+Tet. The positions of molecular weight standards are indicated to the left. Filters were reacted with α-GFP and goat α-mouse secondary conjugated with horseradish peroxidase. The bound complexes were detected with ECL reagents and exposure to x-ray film. C. Identical filters were reacted with α-GFP and goat α-mouse secondary conjugated with alkaline phosphatase. The fluorescent ECF product was visualized with a Molecular Dynamics Storm. The fluorescent image was quantitated using Imagequant. The values were normalized to normal GFP-BLM expression.

The expression of GFP-BLM is inducible in stable transfected BS cells.
A. Equal amounts of total protein (10 μg) from whole cell lysates were resolved on a SDS-polyacrylamide gel and transferred to a membrane. The bound primary α-BLM and goat α-rabbit alkaline phosphatase conjugated secondary antibody complexes were quantitated using a ECF fluorescent alkaline phosphatase substrate. 1) GM0637G normal SV40-transformed fibroblast cells (0.17), 2) HG2522-1a cells + GFP-BLM gene no inducer (0.03), 3) +0.0625 μg/ml Tet (0.05), 4) +0.125 μg/ml Tet (0.11), 5) +0.25 μg/ml Tet (0.16), 6) +0.5 μg/ml Tet (0.3), 7) +1 μg/ml Tet (0.3), 8) +2 μg/ml Tet (0.14). The ECF fluorescent product was visualized directly with a Molecular Dynamics Storm PhosphorImager. B. The fluorescent image was quantitated using Imagequant. The amount of BLM in the lysates was normalized to the amount in equivalent total protein in the GM0637G cells. The data points are the average of two experiments. The standard deviations for each concentration are reported in parentheses above.

Purification and Assay of GFP-BLM. A. Fluorescent microscope image of GFP-BLM in the DAPI-stained nucleus of an Sf9 cell 72 hours after infection with a recombinant baculovirus. GFP-BLM forms large green aggregates. B. Silver-stained SDS 4-12% polyacrylamide gel of the purification of GFP-BLM using Macroprep Q ion exchange chromatography followed by metal chelate resin chromatography. Lane Q shows the ion exchange column pool of the green GFP-BLM fractions and lanes W1-W3 are proteins eluted from the metal resin with increasing imidazole washes (wash1 = 10 mM, wash2 = 50 mM, wash3 = 100 mM). The elution lanes show purified GFP-BLM eluted with a final step of 200 mM imidazole. The molecular weights (MW) of the standard markers (BioRad Precision) are indicated. C. Autoradiogram of a thin-layer plate ATPase assay of GFP-BLM. The amount of protein in the assay is shown. The addition of single-stranded Mpl8 DNA is indicated. Calf intestinal phosphatase (CIP) is used as a positive control for [γ-³²P]ATP hydrolysis. D. Unwinding of a ³²P-labeled 54 base oligonucleotide annealed to Mpl8 DNA by GFP-BLM. The position of the substrate (S) and the product (P) on the autoradiogram of a 10% polyacrylamide gel are indicated. The gel shows reactions containing 25 ng of GFP-BLM incubated for increasing time at 27°C and unwinding by increasing amounts of GFP-BLM incubated at 27°C for 30 minutes.