1. GRACILE Syndrome, a Lethal Metabolic Disorder with Iron Overload, Is Caused by a Point Mutation in BCS1L 
Ilona Visapää, Vineta Fellman, Jouni Vesa, Ayan Dasvarma, Jenna L. Hutton, Vijay Kumar, Gregory S. Payne, Marja Makarow, Rudy Van Coster, Robert W. Taylor, Douglass M. Turnbull, Anu Suomalainen, Leena Peltonen 
The American Journal of Human Genetics 
Volume 71, Issue 4, Pages (October 2002)
DOI: /342773
Copyright © 2002 The American Society of Human Genetics Terms and Conditions

2. Figure 1
A, Critical GRACILE region. The locations of the two functionally and positionally promising candidate genes, BCS1L and ABCB6, are shown in relation to the markers demonstrating linkage disequilibrium in GRACILE chromosomes. The marker order and distances are presented as in the NCBI MapViewer, May B, Genomic structure of the BCS1L gene. The sizes of the exons and introns are indicated in base pairs. The BCS1L mutations reported here (in Finnish and British patients) are indicated above the figure, and the BCS1L mutations described elsewhere (de Lonlay et al. 2001) are marked below. The sizes of the first exon and the first intron (marked with asterisks) vary because of alternative splicing. C, The BCS1L polypeptide of 419 amino acid residues. The numbers indicate the polypeptide regions encoded by distinct exons of the BCS1L gene. Residues 9–32 correspond to the transmembrane region of the yeast BCS1p protein (Fölsch et al. 1996). Residues 224–344 represent the conserved AAA domain, according to CD-Search (Marchler-Bauer et al. 2002) at the NCBI BLAST service.
The American Journal of Human Genetics  , DOI: ( /342773)
Copyright © 2002 The American Society of Human Genetics Terms and Conditions

3. Figure 2
Northern blot analysis of BCS1L in Finnish patients with GRACILE syndrome. For the fibroblast filter, each line was loaded with 9 μg poly-A-RNA; for the liver filter, each line was loaded with 6 μg poly-A-RNA. The same filters were rehybridized with β-actin cDNA, to control for the total mRNA quantity.
The American Journal of Human Genetics  , DOI: ( /342773)
Copyright © 2002 The American Society of Human Genetics Terms and Conditions

4. Figure 3
Subcellular localization of the wild-type and S78G mutant BCS1L polypeptides in transiently transfected COS-1 cells. The BCS1L polypeptides were detected with anti-FLAG antibody (green), and the distribution was compared with mitochondrial staining (red). Colocalization is indicated in yellow. Magnification = 630×.
The American Journal of Human Genetics  , DOI: ( /342773)
Copyright © 2002 The American Society of Human Genetics Terms and Conditions

5. Figure 4
Expression of the wild-type and S78G mutant BCS1L polypeptides. COS-1 cells transfected with the wild-type and mutant BCS1L constructs were subjected to western blot analysis using anti-FLAG antibody. Each lane was loaded with 25 μg protein. pCMV5 = cells transfected with the vector only.
The American Journal of Human Genetics  , DOI: ( /342773)
Copyright © 2002 The American Society of Human Genetics Terms and Conditions

6. Figure 5
Stability of the wild-type and S78G mutant BCS1L polypeptides. Transiently transfected COS-1 cells were pulse-labeled for 1 h and were chased for 0, 12, 24, 36, and 48 h. The BCS1L polypeptides were immunoprecipitated, were separated on 10% SDS-PAGE, and were visualized by fluorography. The radioactive bands were quantitated by densitometry. The results are presented as a percentage of values at 0 h chase. The values on the curve are average values of duplicate experiments, with error bars showing the SD.
The American Journal of Human Genetics  , DOI: ( /342773)
Copyright © 2002 The American Society of Human Genetics Terms and Conditions

7. Figure 6
Yeast complementation experiment with BCS1L. The BCS1 deletion yeast strain was transformed with the wild-type and mutant BCS1L constructs in a high-copy yeast expression plasmid pRS425. The wild-type yeast BCS1 cDNA in the same vector was used as a positive control, and the plain vector was used as a negative control. A, YPEG (ethanol-glycerol) plate incubated at 30°C for 11 d. B, YPEG plate incubated at 37°C for 8 d. C, YPD (glucose) plate incubated at 30°C for 3 d.
The American Journal of Human Genetics  , DOI: ( /342773)
Copyright © 2002 The American Society of Human Genetics Terms and Conditions