Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM-carried genes remains to be clarified. The human colorectal cancer cell line NCI-H716 and human malignant primitive neuroectodermal tumor cell line SK-PN-DW are known to contain many DMs. To examine the amplification of DM-carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI-H716 and SK-PN-DW tumor cells. We identified the amplification regions and the DM-carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM-carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM-carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM-carried genes led to a decrease in the number of DMs and reduced the amplification of the DM-carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of ?H2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM-carried genes resulted in a decrease in cell proliferation and invasion ability.
An insert mutation of YELLOW-GREEN LEAF2 , encoding Heme Oxygenase 1 , results in significant reduction of its transcript levels, and therefore impairs chlorophyll biosynthesis in rice. Heme oxygenase (HO) in higher plants catalyzes the degradation of heme to synthesize phytochrome precursor and its roles conferring the photoperiodic control of flowering in rice have been revealed. However, its involvement in regulating rice chlorophyll (Chl) synthesis is not fully explored. In this study, we isolated a rice mutant named yellow-green leaf 2 (ygl2) from a (60)Co-irradiated population. Normal grown ygl2 seedlings showed yellow-green leaves with reduced contents of Chl and tetrapyrrole intermediates whereas an increase of Chl a/b ratio. Ultrastructural analyses demonstrated grana were poorly stacked in ygl2 mutant, resulting in underdevelopment of chloroplasts. The ygl2 locus was mapped to chromosome 6 and isolated via map-based cloning. Sequence analysis indicated that it encodes the rice HO1 and its identity was verified by transgenic complementation test and RNA interference. A 7-Kb insertion was found in the first exon of YGL2/HO1, resulting in significant reduction of YGL2 expressions in the ygl2 mutant. YGL2 was constitutively expressed in a variety of rice tissues with the highest levels in leaves and regulated by temperature. In addition, we found expression levels of some genes associated with Chl biosynthesis and photosynthesis were concurrently altered in ygl2 mutant. These results provide direct evidence that YGL2 has a vital function in rice Chl biosynthesis.
Double minutes (DMs) are hallmarks of gene amplification. However, their molecular structure and the mechanisms of formation are largely unknown. To elucidate the structure and underlying molecular mechanism of DMs, we obtained and cloned DMs using microdissection; and degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR) from the ovarian cancer cell line UACC-1598. Two large amplicons, the 284 kb AmpMYCN, originating from locus 2p24.3 and the 391 kb AmpEIF5A2, from locus 3q26.2, were found co-amplified on the same DMs. The two amplicons are joined through a complex 7 kb junction DNA sequence. Analysis of the junction has revealed three de novo created small palindromes surrounding the six breakpoints. Consistent with these observations, we further found that 70% of the 57 reported DM junction sequences have de novo creation of small palindromic sequences surrounding the breakpoints. Together, our findings indicate that de novo-generated small palindromic sequences are characteristic of amplicon boundary junctions on DMs. It is possible that the de novo-generated small palindromic sequences, which may be generated through non-homologous end joining in concert with a novel DNA repair machinery, play a common role in amplicon rejoining and gene amplification.
Previously, we identified 3 overlapping regions showing loss of heterozygosity (LOH, R(1)-R(3) from 11 to 30 cM) on chromosome 17 in 45 primary gastric cancers (GCs). The data indicated the presence of tumor suppressor genes (TSGs) on chromosome 17 involved in GC. Among the putative TSGs in these regions, HIC1 (in SR(1)) and TOB1 (in SR(3)) remain to be examined in GC. By immunohistochemistry (IHC), methylation-specific PCR (MSP) and western blot, we evaluated the expression and regulation status for HIC1 and TOB1 protein in GC. We narrowed down the deletion intervals on chromosome 17 and defined five smaller LOH subregions, SR(1)-SR(5) (0.54 to 3.42 cM), in GC. We found that HIC1 had downregulated expression in 86% (91/106) and was methylated in 87% (26/30) of primary GCs. Of the primary GCs showing downregulation of HIC1 protein, 75% (18/24) had methylated HIC1 gene. TOB1 was either absent or expressed at reduced levels in 75% (73/97) of the GC samples. In addition, a general reduction was found in total and the ratio of unphosphorylated to phosphorylated TOB1 protein levels in the differentiated GC cell lines. Further analysis revealed significant simultaneous downregulation of both HIC1 and TOB1 protein in GC tissue microarray samples (67%, 52/78) and in primary GCs (65%, 11/17). These results indicate that silencing of HIC1 and TOB1 expression is a common occurrence in GC and may contribute to the development and progression of the disease.
We conducted a meta-analysis to assess the association between tumor necrosis factor-alpha (TNF-alpha) gene TNFA -308 (G>A), TNFA -238 (G>A), TNFA -857 (C>T), TNFA -863 (C>A), TNFA -1031 (T>C), TNFA -1210 (A>T) polymorphisms and breast cancer(BC) susceptibility. We also performed subgroup analyses based on ethnicity (Caucasian, Asian, and African). An extensive search was performed to identify all case-control studies investigating such association. Thirteen eligible studies, including 10,236 BC patients and 13,143 controls, were identified. No significant association was observed in all genotypes in worldwide populations, but stratification by ethnicity indicated that the TNFA -308 A allele was associated with a decreased risk of BC compared with the G allele in Caucasian individuals (OR = 0.927, 95%CI = 0.879-0.978). Similar results were obtained when the A/A +A/G genotype was compared with the G/G genotype. In addition, meta-analysis results indicated that the A/A genotype of TNFA -308 was a risk factor for BC in African (A/A vs. G/G OR = 4.085 95%CI = 1.460-11.425; A/A vs. G/A OR = 4.861 95%CI = 1.746-13.527; A/A vs. G/A + G/G OR = 4.246 95%CI = 1.551-11.625), but not in Caucasian or Asian individuals. In conclusion, the results of this meta-analysis indicate that the TNFA -308 A allele may be an important protective factor for BC in European individuals, but it is not likely to confer susceptibility to BC in worldwide populations. In addition, the AA genotype of TNFA -308 may be a risk factor for BC in African individuals. Besides, other polymorphisms were not associated with BC susceptibility.
Toll-like receptors (TLRs) are key molecular sensors used by the mammalian innate immune system to detect microorganisms. Although TLR functions in colonic immune homeostasis and tolerance to commensal bacteria have been intensively researched, the precise roles of different TLRs in response to pathogen infection in the gut remain elusive. Peyer patches are the major entrance of Salmonella infection and antigen transportation in intestine. Here, we report that, in contrast to TLR5 as a "carrier of Salmonella," TLR11 works as a "blocker of Salmonella" to prevent highly invasive Salmonella from penetrating into the murine Peyer patches and spreading systemically. TLR11 plays an important role in mediating TNF-? induction and systemic inflammation in response to Salmonella infection. Remarkably, in mice lacking TLR11, apparent hemorrhages at Peyer patches are induced by highly invasive Salmonella, a phenotype resembling human Salmonella infection. Therefore, our results indicate a potentially important role for TLR11 in preventing murine intestinal infection and modulating antigen transportation in the gut and imply an important role for various TLRs in cooperation with tight control of pathogens penetrating into Peyer patches. The TLR11 knock-out mouse can serve as a good animal model to study Salmonella infection.
microRNAs (miRNAs) bind to the 3 untranslated regions (UTRs) of messenger RNAs, where they interfere with translation of genes that regulate cell differentiation, apoptosis and tumourigenesis. The histone methyltransferase SET8 has been reported to methylate TP53 and regulate genomic stability. We analysed a single nucleotide polymorphism (rs16917496) within the miR-502 miRNA seed region at the 3 UTR of SET8 in small-cell lung cancer (SCLC) patients. The SET8 CC+CT genotype was identified to be independently associated with longer survival in SCLC patients by multivariate analysis (relative risk, 0.453; 95% CI 0.217-0.944; p=0.035). The analysis of genetic polymorphisms in miRNA binding sites may help to identify patient subgroups at high risk of poor outcome.
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