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

MOESM3 of A new genome-scale metabolic model of Corynebacterium glutamicum and its application

Additional file 3. The reactions and metabolites of iCW773

MOESM2 of A RecET-assisted CRISPR–Cas9 genome editing in Corynebacterium glutamicum

Additional file 2: Standard protocol of a RecET-assisted CRISPR-Cas9 genome editing in Corynebacterium glutamicum

MOESM1 of A RecET-assisted CRISPR–Cas9 genome editing in Corynebacterium glutamicum

Additional file 1: Figure S1. Colony PCR verification of cas9 integration. Figure S2. Colony PCR verification of upp deletion in WT::P hom -cas9. Figure S3. Colony PCR verification of upp deletion in WT::P tuf -cas9. Figure S4. Colony PCR verification of upp deletion in WT::P tuf -rbs1-cas9. Figure S5. Colony PCR verification of upp deletion in WT::P tuf -rbs2-cas9. Figure S6. Colony PCR verification of recET integration. Figure S7. Colony PCR verification of upp deletion in WT::P tuf -rbs2-cas9::P prp -recET. Figure S8. Colony PCR verification of upp deletion in WT::P tuf -rbs2-cas9::P prp -rbs3-recET. Figure S9. Colony PCR verification of upp deletion in WT::P tuf -rbs2-cas9::P prp -rbs4-recET (EDT). Figure S10. Colony PCR verification of argR deletion in EDT. Figure S11. Colony PCR verification of farR deletion in EDT△argR. Figure S12. Colony PCR verification of ldh deletion via pHA500sgRNA ldh in EDT. Figure S13. Colony PCR verification of ldh deletion via pHA1000sgRNA ldh in EDT. Figure S14. Colony PCR verification of 1-kb fragment deletion at the CGP3 locus in EDT. Figure S15. Colony PCR verification of 10-kb fragment deletion at the CGP3 locus in EDT. Figure S16. Colony PCR verification of 20-kb fragment deletion at the CGP3 locus in EDT. Figure S17. Colony PCR verification of gfp and hom-thrB insertion at the upp locus in EDT. Figure S18. Colony PCR verification of gfp insertion at the CGP1 locus in ET. Figure S19. Colony PCR verification of gfp insertion at the CGP2 locus in EDT. Figure S20. Colony PCR verification of gfp insertion at the CGP3 locus in EDT. Figure S21. Colony PCR verification of P tuf -hom-thrB, P tuf -hom-thrB-P glyA -lysC-thrC and P tuf -trpEG-P glyA - trpDC-P sod -trpBA insertions at the upp locus in EDT. Figure S22. Colony PCR verification of lacZ fragment insertion into the genomic locus between cgl0900 and cgl0901. Figure S23. Colony PCR verification of ldh deletion in EDT. Figure S24. Colony PCR verification of hdpA deletion in EDT△ldh. Figure S25. Colony PCR verification of plug out of cas9–recET expression cassette in EDT△ldh△hdpAP hom -pgk. Table S1. Editing efficiencies using different genome editing methods in C. glutamicum. Table S2. Strains used in this study. Table S3. Plasmids used in this study. Table S4. Primers used in this study

Reprogramming One-Carbon Metabolic Pathways To Decouple l‑Serine Catabolism from Cell Growth in <i>Corynebacterium glutamicum</i>

l-Serine, the principal one-carbon source for DNA biosynthesis, is difficult for microorganisms to accumulate due to the coupling of l-serine catabolism and microbial growth. Here, we reprogrammed the one-carbon unit metabolic pathways in <i>Corynebacterium glutamicum</i> to decouple l-serine catabolism from cell growth. <i>In silico</i> model-based simulation showed a negative influence on <i>glyA</i>-encoding serine hydroxymethyltransferase flux with l-serine productivity. Attenuation of <i>glyA</i> transcription resulted in increased l-serine accumulation, and a decrease in purine pools, poor growth and longer cell shapes. The <i>gcvTHP</i>-encoded glycine cleavage (Gcv) system from <i>Escherichia coli</i> was introduced into <i>C. glutamicum</i>, allowing glycine-derived ¹³CH₂ to be assimilated into intracellular purine synthesis, which resulted in an increased amount of one-carbon units. Gcv introduction not only restored cell viability and morphology but also increased l-serine accumulation. Moreover, comparative proteomic analysis indicated that abundance changes of the enzymes involved in one-carbon unit cycles might be responsible for maintaining one-carbon unit homeostasis. Reprogramming of the one-carbon metabolic pathways allowed cells to reach a comparable growth rate to accumulate 13.21 g/L l-serine by fed-batch fermentation in minimal medium. This novel strategy provides new insights into the regulation of cellular properties and essential metabolite accumulation by introducing an extrinsic pathway

A Novel Tool for Microbial Genome Editing Using the Restriction-Modification System

Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, <i>bceSIIM</i> (RMGE-<i>bceSIIM</i>), as the counter-selection cassette was developed to edit the genome of <i>Escherichia coli</i>. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-<i>mcrA</i>) was established in <i>Bacillus subtilis</i>. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in <i>E. coli</i> using RMGE-<i>bceSIIM</i> to introduce a single base mutation of A128C into the <i>rpsL</i> gene. In addition, the RMGE-<i>mcrA</i> technique was applied to delete the <i>CAN1</i> gene in <i>Saccharomyces cerevisiae</i> DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in <i>E. coli</i>, <i>B. subtilis</i>, and <i>S. cerevisiae</i> suggests the potential universal usefulness of this technique for microbial genome manipulation

Bacterial Genome Editing via a Designed Toxin–Antitoxin Cassette

Manipulating the bacterial genomes in an efficient manner is essential to biological and biotechnological research. Here, we reprogrammed the bacterial TA systems as the toxin counter-selectable cassette regulated by an antitoxin switch (TCCRAS) for genetic modifications in the extensively studied and utilized Gram-positive bacteria, <i>B. subtilis</i> and <i>Corynebacterium glutamicum</i>. In the five characterized type II TA systems, the RelBE complex can specifically and efficiently regulate cell growth and death by the conditionally controlled antitoxin RelB switch, thereby serving as a novel counter-selectable cassette to establish the TCCRAS system. Using a single vector, such a system has been employed to perform in-frame deletion, functional knock-in, gene replacement, precise point mutation, large-scale insertion, and especially, deletion of the fragments up to 194.9 kb in <i>B. subtilis</i>. In addition, the biosynthesis of lycopene was first achieved in <i>B. subtilis</i> using TCCRAS to integrate a 5.4-kb fusion cluster (<i>P</i>_<i>spac</i>–<i>crtI</i>–<i>crtE</i>–<i>crtB</i>). The system was further adapted for gene knockdown and replacement, and large-scale deletion of the fragments up to 179.8 kb in <i>C. glutamicum</i>, with the mutation efficiencies increased by 0.8–1.0-fold compared to the conventional SacB method. TCCRAS thus holds promise as an effective and versatile genome-scale engineering technology for metabolic engineering and synthetic genomics research in a broad range of the Gram-positive bacteria

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

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

Calculated LOD scores for markers on rat chromosome 12.

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