File S1 - Mapping of Mcs30, a New Mammary Carcinoma Susceptibility Quantitative Trait Locus (QTL30) on Rat Chromosome 12: Identification of Fry as a Candidate Mcs Gene

Contains: Table S1. Complete list of Rat Simple Tandem Repeat (STR) Markers used for low-density linkage analysis. Table S2.Coverage of the genome (autosomes) by STR markers used in low-density linkage analysis. Table S3. Primers used to generate amplicons and sequence the Brca2 gene from F344 and Cop rat strains. Table S4. Primers used to generate amplicons and sequence the Rat Fry gene from F344 and Cop rat strains. Table S5. Genbank Accession numbers for all genes sequenced in this study. Figure S1. Histogram showing the incidence (frequency on left axis) of mammary tumors detected in N2 backcross progeny on each of the indicated days post exposure to NMU. The incidence mammary tumors (left axis) and percent of animals with tumors (right axis) as a function of time after exposure is indicated by the green dotted line. Figure S2. Schematic representation of the contig comprising overlapping Bacterial Artificial Chromosome (BACs) containing rat genomic DNA assembled using the DNA sequence of the D12Rat59 STR marker as the seed (not drawn to scale). Rat BAC clones encompassing particular STR markers were obtained from the Children’s Hospital Oakland Research Institute (CHORI-230 BAC library) and verified by PCR amplification to contain the STR sequence. R and F refer to the forward and reverse orientation. Vertical lines indicate sequence overlap. Figure S3. Metaphase chromosome spreads prepared from mouse embryo fibroblast fibroblasts isolated from (F344 X Cop)F1 progeny were hybridized with Bacterial Artificial Chromosomes containing STR markers that comprise the Mcs30 locus or genes on RNO12 used as hybridization controls (see Table 2). Hybridization signals are pseudo-colored red for clarity, and DAPI banding is displayed as grey values. BAC 1: CH230-152N10 containing D12Rat1 hybridized to 12q11-12. BAC 2: CH230-381M14 containing D12Rat59 hybridized to 12q11-12. BAC 3: CH230-275K13 containing D12Rat59 hybridized to 12q11-12. BAC 4: CH230-85G15O containing D12Rat59 hybridized to 12q11-12. BAC 5: CH230-151L24 containing the Epo gene hybridized to 12q11-12. BAC 6: CH230-123F8 containing the PAI 1A1 gene hybridized to 12q11-12. Figure S4. Relative mRNA expression levels of candidate genes from the Mcs30 locus on Chromosome 12 in normal rat mammary tissue before and after NMU exposure. (A). RNA was isolated from normal mammary tissue of 55 day-old female Fischer 344 and Copenhagen rats, before and 6 h, one day and 30 days after exposure exposing female F344 and Copenhagen rats at to a carcinogenic dose of NMU. Gene expression levels of Insr and Stard13 on chromosome 12 were compared by semi-quantification PCR analysis. Expression levels were normalized to expression of beta-actin mRNA; (B). Image densitometry in (B) was quantified using Image J software. (DOC)