MASALAH-MASALAH PEMBELAJARAN YANG DIHADAPI WIDYAISWARA : Studi Kasus Pada Lembaga Diktat Pemda Tk.I Propinsi Bengkulu

<div><p>Rat strains differ dramatically in their susceptibility to mammary carcinogenesis. On the assumption that susceptibility genes are conserved across mammalian species and hence inform human carcinogenesis, numerous investigators have used genetic linkage studies in rats to identify genes responsible for differential susceptibility to carcinogenesis. Using a genetic backcross between the resistant Copenhagen (Cop) and susceptible Fischer 344 (F344) strains, we mapped a novel mammary carcinoma susceptibility (<i>Mcs30</i>) locus to the centromeric region on chromosome 12 (LOD score of ∼8.6 at the D12Rat59 marker). The <i>Mcs30</i> locus comprises approximately 12 Mbp on the long arm of rat RNO12 whose synteny is conserved on human chromosome 13q12 to 13q13. After analyzing numerous genes comprising this locus, we identified <i>Fry</i>, the rat ortholog of the furry gene of <i>Drosophila melanogaster,</i> as a candidate <i>Mcs</i> gene. We cloned and determined the complete nucleotide sequence of the 13 kbp <i>Fry</i> mRNA. Sequence analysis indicated that the <i>Fry</i> gene was highly conserved across evolution, with 90% similarity of the predicted amino acid sequence among eutherian mammals. Comparison of the <i>Fry</i> sequence in the Cop and F344 strains identified two non-synonymous single nucleotide polymorphisms (SNPs), one of which creates a putative, de novo phosphorylation site. Further analysis showed that the expression of the <i>Fry</i> gene is reduced in a majority of rat mammary tumors. Our results also suggested that FRY activity was reduced in human breast carcinoma cell lines as a result of reduced levels or mutation. This study is the first to identify the <i>Fry</i> gene as a candidate <i>Mcs</i> gene. Our data suggest that the SNPs within the <i>Fry</i> gene contribute to the genetic susceptibility of the F344 rat strain to mammary carcinogenesis. These results provide the foundation for analyzing the role of the human <i>FRY</i> gene in cancer susceptibility and progression.</p></div

FISH mapping of BAC CH230-85G15, containing D12RAT59, to rat chromosome 12q11-q12.

<p>Metaphase chromosome spreads were prepared from skin fibroblasts isolated from the BN/SsNHsdMcw rat strain and from cultured embryo fibroblast isolated from (F433 X Cop) F1 progeny rats. Metaphase chromosomes were hybridized with fluorescently-labeled BAC CH230-85G15, which contains D12RAT59, the marker on RNO12 showing tightest linkage to <i>Mcs30</i>. The hybridization signals are pseudo-colored red for clarity, and DAPI banding is displayed as either grey or white values. Other BACs containing D12Rat59 were also hybridized to 12q11-12 (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070930#pone-0070930-t002" target="_blank">Table 2</a> and Figure S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070930#pone.0070930.s001" target="_blank">File S1</a>).</p

Ideogram of rat Chromosome 12 (RNO12).

<p>Ideogram shows the relative locations (drawn to scale) and nucleotide positions (in brackets) for STR markers (blue bars and font) and genes (red bars and bold black font). The placement of genes and markers is based on the draft rat genome sequence (RGD Build 5.1 Assembly (Annotation Release103)) The green bar to the left indicates the position of the 11 Mbp <i>Mcs30</i> locus (QTL30). The position of the centromere is proposed on the basis of FISH mapping, which places D12 Rat1 on RNO12p11. <i>* Although D12Rat1 is also present on RNO1 (RGD Build 5.1 Assembly (Annotation Release103)), the sequence on RON1 is later significantly shorter (93bp) than the sequence on RNO12 (137 bp). Variants of the latter were used for mapping.</i></p

Calculated LOD scores for markers on rat chromosome 12.

<p>Note: LOD, logarithm of the odds ratio.</p

Alignment of Proteins Encoded by <i>FRY</i> gene orthologs from different species.

<p>The <i>Fry</i> allele present in the F344 allele carries two non-synonymous SNPS at amino acid residues, codon 661(Panel A) and 2170 (Panel B), are conserved in a majority of FRY orthologs (showing part of aligned results due to space limit). Strain specific SNPs present in the Fischer 344 rat strain replaces Aspartic Acid (D) and Alanine(A) residues present in Cop rat with amino acid Glutamic Acid (E) and Serine(S) in F344 rat, at amino acid 661 and 2170, respectively.</p

Prediction of a novel phosphorylation site in the F344 Fry allele using NetPhos2.0 phosphorylation prediction software.

<p>Prediction of a novel phosphorylation site in the F344 Fry allele using NetPhos2.0 phosphorylation prediction software.</p

Interval mapping of putative quantitative trait loci (QTL) on rat chromosomes 12 that confer susceptibility to NMU-induced mammary carcinogenesis.

<p>The genetic map positions of polymorphic STR markers are indicated by hatch marks and are designated to the left. Genetic distances between markers in centiMorgans are drawn to scale within each panel. The calculated Likelihood Ration Statistic (LRS) values for linkage of the Quantitative trait (measured as log of tumor numbers) within chromosomal intervals are plotted as X to the right (black line). <b>A)</b> Linkage calculated on chromosome 12, using 99 animals genotyped at the markers indicated on the left. <b>B)</b> Linkage on chromosome 12, as calculated using 324 animals genotyped at the markers indicated on the left. The vertical green lines indicate the suggestive (LRS = 2.7 for Panels A; LRS = 1.9 for Panel B), significant (LRS = 8.0 for Panels A; LRS = 7.2 for Panels B), and highly significant (LRS = 15.0 for Panels A; LRS = 13.4 for Panels B) thresholds as calculated by permutation studies. The additive effect functions are represented as dotted red lines, with negative values to the left and positive values to the right.</p

Comparison of <i>Brca2</i> mRNA levels in mammary tissue and DNA sequences in Cop and F344 rats.

<p>(A) Steady-state levels of the 13 kb Brca2 mRNA were compared by Northern blot analysis. <i>Brca2</i> mRNA was expressed at comparable levels in both strains. (B) Comparison of <i>Brca2</i> cDNA sequences in Cop and F344 rat strains. DNA sequence analysis detected two synonymous single nucleotide polymorphisms (SNP); a T to C transition in Exon11 and C to T transition in Exon25.</p

BACs used for FISH. BACS containing sequences from the D12Rat1, D12Rat59, Epo and PAI1A1were used from physical mapping of loci to Rat chromosome 12.

<p>BACs used for FISH. BACS containing sequences from the D12Rat1, D12Rat59, Epo and PAI1A1were used from physical mapping of loci to Rat chromosome 12.</p

Expression of rat <i>Fry</i> mRNA in rat tissues and mammary tumors.

<p>Semi-quantitative RT-PCR analysis of steady-state <i>Fry</i> mRNA levels in somatic tissues of Cop and F344 rats. β-actin expression was used for normalization (lower panel). (<b>B</b>) Semi-quantitative RT-PCR analysis of steady-state Fry mRNA levels in normal mammary tissue, mammary tumors and normal mammary tissue collected from the matched tumor bearing F344 rats. β-actin mRNA of rat was used for normalization (lower panel). (C) Image densitometry in (B) was quantified using Image J software.</p