Project description:Gene amplification, copy-number increases of particular genes and surrounding genomic segments, promotes cancer progression and acquired therapy resistance. Thus, understanding genetic traits that confer gene amplification proficiency is important. The primary step for gene amplification is spontaneous DNA rearrangements initiated by DNA breaks. Here we show that mammalian cells became gene amplification-proficient when we knocked down Mre11/Rad50/Nbs1 (MRN) complex, a multifunctional complex that guards the genome from DNA breaks. Cells with reduced Mre11 experienced severe replication stress, with marked increases of single-stranded breaks followed by double-stranded breaks during DNA replication. Such breaks underlay for the increase in spontaneous gene amplification. Other traits associated with replication stress, such as impaired intra-S phase checkpoint and global transcriptional changes in DNA metabolism genes also contributed to gene amplification proficiency. Our results define Mre11 deficiency as a cause of replication stress and gene amplification proficiency and provide a candidate marker for aggressive cancer phenotypes. We sequenced four samples: 2 control, GFP-expressing samples and 2 Mre11 knockdown cells
Project description:Microarray analysis of E. amylovora treated with compounds no. 3 and no. 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were, respectively, induced and suppressed by both compounds as compared to DMSO control. Majority of T3SS genes in E. amylovora including hrpL and avrRpt2 effector gene were suppressed by both compounds. Compound no. 3 also suppressed the transcription of amylovoran precursor and biosynthesis genes. On the other hand, both compounds significantly induced expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein encoding genes were also activated by both compounds. Erwinia amylovora WT strain treated with compound No. 3 and No. 9 and compared to DMSO control. Four biological replicates for each chemical treatment (two samples were combined) were hybridized to three arrays. Four biological replicates (two each combined) for DMSO treatment were hybridized to two arrays.
Project description:We conducted genome-wide microarray analyses to determine the regulons of RcsB and RcsC in liquid medium and, for the first time, on immature pear fruit. Our array analyses identified a total of 648 genes differentially regulated by the RcsCB in vitro and in vivo. Consistent with our previous findings, RcsB acts as a positive regulator in both conditions, while RcsC positively controls amylovoran biosynthetic gene expression in vivo, but negatively in vitro. Besides amylovoran biosynthesis and regulatory genes, cell wall and cell envelope (membrane) as well as regulatory genes were the major components of the RcsBC regulon, including many novel genes. In addition, we have also demonstrated that transcripts of rcsA, rcsC and rcsD genes, but not rcsB gene, were up-regulated when grown in minimal medium or after infection of pear fruits compared to LB medium. Furthermore, a hidden Markov model (HMM) has predicted 60 genes with candidate RcsB binding site in the intergenic regions of the E. amylovora ATCC 49946 genome and 18 (28) of them were identified in the microarray assay. Based on our findings, a working model has been proposed to illustrate how the Rcs phosphorelay system regulates virulence gene expression in E. amylovora. A total of 12 samples were analyzed in two conditions: For in vitro condition (MBMA medium + 1% sorbitol), Erwinia amylovora wild type strain (2 replicates); E. amylovora rcsB mutant strain (2 replicates); E. amylovora rcsC mutant strain (2 replicates): For in vivo condition (on wounded immature pear fruits), Erwinia amylovora wild type strain (2 replicates); E. amylovora rcsB mutant strain (2 replicates); E. amylovora rcsC mutant strain (2 replicates).
Project description:Microarray analysis was used to identify genes that were controlled by AmyR in minimal and on immature pear fruits. Consistent with amylovoran production, an inverse correlation was observed between amyR expression and the expression level of amylovoran biosynthetic genes in liquid media. Interestingly, over-expression of AmyR suppressed the expression of type III secretion system genes including hrpA, hrpN and dspEF after pear fruit infection. Consistent with levan production and swarming motility, levasucrase and flagellar genes were both down-regulated both in the amyR mutant and over-expression strains in liquid media. Together, our results suggest that AmyR plays an important role in regulating bacterial exopolysaccharide production and virulence in E. amylovora. A total of 14 samples were analyzed in two conditions: For the in vitro condition (MBMA medium + 1% sorbitol), Erwinia amylovora wild type strain (2 replicates); E. amylovora amyR mutant strain (3 replicates); E. amylovora amyR over-expression strain (3 replicates); For the in vivo condition (on wounded immature pear fruits), Erwinia amylovora wild type strain (2 replicates); E. amylovora amyR mutant strain (2 replicates); E. amylovora amyR over-expression strain (2 replicates).
Project description:To extract the differentially expressed miRNA between control vs diabetic mice skeletal muscle. We used obese diabetic (C57BL/KsJ db/db) and normal control (C57BL/KsJ db+) mice, which were obtained from the Animal House Facility of the Central Drug Research Institute (CSIR), Lucknow, India.