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

Binding equilibrium for aptamers and their HF cell epitopes.

<p>Fluorescence quantification of FAM-labeled aptamers bound to HF cells. Aptamer binding curves (line and data points) were generated by graphing the average fluorescence with the error bars representing the standard error of the mean for each data point (n = 4 per data point). These data were fitted to the model Y = Bmax*X(Kd+X) (solid line) which was generated using nonlinear regression analysis for one site specific binding: aptamer 13 (K_d = 2.5±0.5 nM); aptamer 14 (K_d = 7.1±0.4 nM); aptamer 20 (K_d = 1.6±0.4 nM); and aptamer 28 (K_d = 6.9±0.2 nM).</p

Predicted aptamer secondary structures.

<p>The secondary structures for each of the aptamers investigated. The structure(s) with the lowest free energy (dG) are presented. Secondary structure predictions were determined using UNAfold software and are sir_graph ® output <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036103#pone.0036103-Zuker1" target="_blank">[13]</a>.</p

Schematic of Adherent Cell-SELEX (AC-SELEX) Procedure.

<p>A starting library of >10¹⁵ aptamers was utilized and eighteen rounds of AC-SELEX were carried out to purify and amplify the aptamers which recognized epitopes present on HF cells and not present on HeLa cells. The starting library was first incubated with the HeLa cell line and the unbound aptamers were recovered and utilized for the AC-SELEX procedure.</p

Aptamer sequences.

<p>All Aptamer sequences are flanked by the primer sequences: 5′ – ATA CCA GCT TAT TCA ATT – N₅₂ - AGA TAG TAA GTG CAA TCT – 3′. Aptamers in <b>bold</b> were randomly chosen for further characterization.</p

The nontumorigenic, HA and tumorigenic SiHa cervical cancer cells after aptamer incubation.

<p>Images depicting the non-transformed revertant, HA cell line and the HPV-transformed cervical cancer cell line, SiHa, after incubation with FAM labeled aptamers. The aptamers generated to target the non-transformed revertant, HF cell line also recognize the non-transformed revertant, HA cell line. Also notice that these aptamers do not recognize epitopes on the SiHa cervical cancer cell line, similar to the results we observed with the HeLa cervical cancer cell line. These results are consistent with the hypothesis that these aptamers are recognizing epitopes that are lost as a result of transformation of cervical epithelial cells by HPV. All images are 40× and are of cells within 24 hours after incubation with 2000 nM aptamers and subsequent fixation and coverslipping with Aquamount.</p

The target (HF) and non-target (HeLa) cell lines after incubation with FAM labeled aptamers.

<p>Images of HF and HeLa cells incubated with fluorescently tagged aptamers. The aptamers are specific for the non-tumorigenic HF cell line. They recognize epitopes on or in the HF cells and do not recognize their binding sites on the tumorigenic HeLa cells. All images are 40× and are of cells within 24 hours after incubation with 2000 nM aptamers and subsequent fixation and coverslipping with Aquamount.</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

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

Calculated LOD scores for markers on rat chromosome 12.

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