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Blood, Vol. 112, Issue 5, 1582-1592, September 1, 2008
Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia
Blood Farrar et al.
112: 1582
Supplemental materials for: Farrar et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 1.3 MB)
- Table S1. RPL35A PCR primers (PDF, 45.7 KB)
- Table S2. Frequency distribution of the number of genes located on all chromosomes, chromosome 3 and chromosome 3q28-ter* (PDF, 12.5 KB)
- Table S3. Candidate 3q28 interval genes selected as under-expressed (PDF, 68.9 KB)
- Figure S1. Design of RPL35A shRNAs (JPG, 44 KB)
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shRNA expressed from a lentiviral vector targeting 4 regions of RPL35A are shown as black bars in relation to the spliced transcript and protein coding region. The effector shRNA sequences are depicted below as hairpins. Nucleotides in black target RPL35A at the indicated locations while gray nucleotides denote loop sequence.
- Figure S2. Illustration of the filtering algorithm implemented to obtain a chromosome 3 or chromosome 3q28-ter candidate gene list (JPG, 25.7 KB)
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Numbers refer to the probes selected as candidate under-expressed in Deletion 1 EBV relative to control EBV cell lines.
- Figure S3. Haploinsufficient RPL35A transcript expression in 3q deletion DBA EBV cell lines (JPG, 76.2 KB)
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Quantitative PCR for RPL35A shows significantly decreased expression in EBV transformed lymphoblasts from two patients with DBA and terminal 3q deletions (Red). EBV cell lines from normal individuals (Blue), two patients with DBA without chromosome 3q deletions or RPS19 mutations (Green) and two patients with DBA characterized by RPS19 mutations (Black) show similar expression of RPL35A. * p < 0.01, **p < 0.001.
- Figure S4. Southern blot localization of 3q deletions 1 and 2 (JPG, 129 KB)
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(A) Genomic DNA was digested with the indicated enzymes, transferred and probed with an LPP intron 7 probe. The Bgl II digestion resulted in an abnormal fragment smaller than the wild-type fragement (red arrow). This fragment was amplified by inverse PCR (B), cloned and sequenced to identify the breakpoint juxtaposed to human  satellite repeat sequence (C). (D) Southern blotting of Deletion 2 with a probe to the intergenic region between HES1 and CPN2 similarly confirmed this breakpoint region. (e) A genomic map of restriction sites and probes used to define these deletions illustrates selected predicted restriction fragments reactive with probes used to define the respective deletions. The precise location of the Deletion 2 breakpoint was not defined but is expected to lie within the 8.1 Kb region defined by the Eco RI fragment displayed. The genomic region displayed is indicated under each figure. For Deletion 2, the interval shown crosses a gap between two contigs estimated at 20 Kb (NCBI Build 36.1).
- Figure S5. A 7S precursor to 5.8S rRNA (JPG, 55.7 KB)
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RNA from UT-7/Epo, EBV and TF-1 cells was analyzed by Northern analysis with the probes indicated above each panel. Boxed panels denote probing from a single blot. The rRNA species are indicated at left. A band migrating just above 5.8S rRNA at a predicted size of ∼325 base pairs was visible using a 5′ITS1 probe in all cells (UT7/Epo shown) as well as with a 5.8S probe (EBV LCL and TF-1 shown; this band is not apparent in UT-7/Epo due to a shorter exposure) but was not seen with a 3′ITS1 probe (EBV LCL), implying a 3′ truncated intermediate between 12 and 5.8S which we have designated “7S.”.
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