The dichotomy of p53 regulation by noncoding RNAs

The p53 tumor suppressor gene is the most frequently mutated gene in cancer. Significant progress has been made to discern the im- portance of p53 in coordinating cellular responses to DNA damage, oncogene activation, and other stresses. Noncoding RNAs are RNA molecules functioning without being translated into proteins. In this work, we discuss the dichotomy of p53 regulation by noncoding RNAs with four unconventional questions. First, is overexpression of microRNAs responsible for p53 inactivation in the absence of p53 mutation？ Second, are there somatic mutations in the noncoding regions of the p53 gene？ Third, is there a germline mutant in the non- coding regions of the p53 gene that predisposes carriers to cancer？ Fourth, can p53 activation mediated by a noncoding RNA mutation cause cancer？ This work highUghts the prominence of noncoding RNAs in p53 dysregutation and tumorigenesis.     

Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms.Driven by a given tissue-specific Gal4,expressing UAS-transgene or UAS-RNAi(RNA interference)could be used to up-or down-regulate target gene expression,respectively.However,the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock.Here,we describe a simple way to modulate optomotor blind(omb)expression levels in its endogenous expression region of the wing disc.We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch.The repression effect is more sensitive to temperature than that of overexpression.At low temperature,overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi.In contrast,at high temperature RNAi predominates in gene expression regulation.By this strategy,we could manipulate omb expression levels at a moderate level.It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.     

The Chlamydomonas Chloroplast HLP Protein Is Required for Nucleoid Organization and Genome Maintenance

The chloroplasts genome （plastome） occurs at high copy numbers per cell. Several chloroplast genome copies are densely packed into nucleoprotein particles called nucleoids. How genome packaging occurs and which proteins organize chloroplast nucleoids are largely unknown. Here, we have analyzed the Chlamydornonas reinhardtii homolog of the bacterial architectural DNA-binding protein HU, the histone-like protein HLP. We show that the Chlarnydornonas HLP protein is targeted to chloroplasts and associates with nucleoids. Knockdown of HLP gene expression by RNA interference （RNAi） alters the structure of chloroplast nucleoids and appears to reduce the level of compaction of chloroplast DNA. Unexpectedly, also chloroplast genome copy numbers are significantly decreased in the RNAi strains, suggesting that, in addition to its architectural role in nucleoid formation, the HIP protein is also involved in chloroplast genome maintenance.     

dsRNA binding protein PACT/RAX in gene silencing,development and diseases

PACT （Protein kinase, interferon-inducible double stranded RNA dependent activator） and its murine ortholog RAX （PKR-associated protein X） were originally identified as a protein activator for the dsRNA-dependent, interferon-inducible protein kinase （PKR）. Endogenous PACT/RAX activates PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. PACT/RAX heterodimerized with PKR and activated it with its third motif in the absence of dsRNA. The activation of PKR leads to enhanced eIF2a phosphorylation followed by apoptosis or inhibition of growth. Besides the role of activating PKR, PACT is associated with a ~500 kDa complex that contains Dicer, hAgo2, and TRBP （TAR RNA binding protein） and it associates with Dicer to facilitate the production of small interfering RNA. PACT/RAX plays an important role in diverse physiological and pathological processes. Pact^-/- mice exhibit notable developmental abnormalities including microtia, with craniofacial ear, and hearing defects. Pact^-/- mice had smaller body sizes and fertility defects, both of which were caused by defective pituitary functions. It was found that dRAX disrupted fly embryos homozygous, displayed highly abnormal commissural axon structure of the central nervous system, and 70% of the flies homozygous for the mutant allele died prior to adulthood. Using high density SNP genotyping arrays, it was found that a mutation in PRKRA （the PACT/RAX gene） is the causative genetic mutation in DYT16, a novel autosomal recessive dystonia-parkinsonism syndrome in Brazilian patients.     

Study on gene sensor based on primer extension

Based on the fact that the resonant frequency of a piezoelectric crystal is the function of its surface deposit, and that the primer extends after it hybridizes with the template, the primer extension gene sensor technique was developed. The prominent feature of the technique is that fast and sensitive frequency signals are used as the monitoring system of gene hybridization and primer strand extension. Results show that this technique may be used in homologous analysis of nucleic acid, trace DNA detection, and determining the integration of DNA. It may also be used for isolation of target gene, gene mutation analysis, and predicting the location of a gene in its genome.     

Germline silencing of UASt depends on the piRNA pathway

正The success of the fruit fly Drosophila melanogaster as a model organism is heavily attributed to the expansive range and multitude of genetic and molecular tools available to modify gene expression at will.The Gal4/UAS binary system is one of the most important and widely used genetic tools in Drosophila designed for targeted gene expression(Brand and Perrimon,1993),which allows ectopic expression of any gene(or transgene)in specific tissues,independent of their native regulators.     

Somatic gene targeting with RNA/DNA chimeric oligonucleotides: an analysis with a sensitive reporter mouse system

BACKGROUND: Targeted gene correction provides a potentially powerful method for gene therapy. RNA/DNA chimeric oligonucleotides were reported to be able to correct a point mutation with a high efficiency in cultured rodent cells, in the body of mice and rats, and in plants. The efficiency of correction in the liver of rats was claimed to be as high as 20% after tail-vein injection. However, several laboratories have failed to reproduce the high efficiency. METHODS: In order to sensitively detect and measure sequence changes by the chimeric oligonucleotides, we used Muta Mouse, a transgenic mouse system for mutation detection in vivo. It carries, on its chromosome, multiple copies of the lambda phage genome with the lacZ(+) gene. Two chimeric oligonucleotides were designed to make a point mutation at the active site of the LacZ gene product. They were injected into the liver with HVJ liposomes, which were demonstrated to allow reliable gene delivery. One week later, DNA was extracted from the liver, and lambda::lacZ particles were recovered by in vitro packaging. The lacZ-negative phage was detected by selection with phenyl-beta-D-galactoside. RESULTS: The mutant frequency of the injected mice was at the same level as the control mouse (approximately 1/10000). Our further restriction analysis and sequencing did not detect the designed mutations. CONCLUSIONS: Gene correction frequency in mouse liver by these oligonucleotides was shown to be less than 1/20000 in our assay with the Muta Mouse system.     

A plausible mechanism for gene correction by chimeric oligonucleotides

Self-complementary chimeric oligonucleotides that consist of DNA and 2'-O-methyl RNA nucleotides arranged in a double-hairpin configuration can elicit a point mutation when targeted to a gene sequence. We have used a series of structurally diverse chimeric oligonucleotides to correct a mutant neomycin phosphotransferase gene in a human cell-free extract. Analysis of structure-activity relationships demonstrates that the DNA strand of the chimeric oligonucleotide acts as a template for high-fidelity gene correction when one of its bases is mismatched to the targeted gene. By contrast, the chimeric strand of the oligonucleotide does not function as a template for gene repair. Instead, it appears to augment the frequency of gene correction by facilitating complex formation with the target. In the presence of RecA protein, each strand of a chimeric oligonucleotide can hybridize with double-stranded DNA to form a complement-stabilized D-loop. This reaction, which may take place by reciprocal four-strand exchange, is not observed with oligonucleotides that lack 2'-O-methyl RNA segments. Preliminary sequencing data suggest that complement-stabilized D-loops may be weakly mutagenic. If so, a low level of random mutagenesis in the vicinity of the chimera binding site may accompany gene repair.     

Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides

Maize plants resistant to imidazolinone herbicides were engineered through targeted modification of endogenous genes using chimeric RNA/DNA oligonucleotides. A precise single-point mutation was introduced into genes encoding acetohydroxyacid synthase (AHAS), at a position known to confer imidazolinone resistance. Phenotypically normal plants from the converted events (C0) were regenerated from resistant calli and grown to maturity. Herbicide leaf painting confirmed the resistance phenotype in C0 plants and demonstrated the anticipated segregation pattern in C1 progeny. DNA cloning and sequencing of the targeted region in resistant calli and derived C0 and C1 plants confirmed the expected mutation. These results demonstrate that oligonucleotide-mediated gene manipulation can be applied to crop improvement. This approach does not involve genomic integration of transgenes. Since the new trait is obtained through modifying a gene within its normal chromosomal context, position effects, transgene silencing, or other concerns that arise as part of developing transgenic events are avoided.     

Optimizing the delivery systems of chimeric RNA.DNA oligonucleotides.

Special oligonucleotides for targeted gene correction have attracted increasing attention recently, one of which is the chimeric RNA.DNA oligonucleotide (RDO) system. RDOs for targeted gene correction were first designed in 1996, and are typically 68 nucleotides in length including continuous RNA and DNA sequences (RNA is 2'-O-methyl-modified). They have a 25 bp double stranded region homologous to the targeted gene, two hairpin ends of T loop and a 5 bp GC clamp, that give the molecule much greater stability [Fig. 1]. One mismatch site in the middle of the double-stranded region is designed for targeted gene therapy. RDOs have been used recently for targeted gene correction of point mutations both in vitro and in vivo, but many problems must be solved before clinical application. One of the solutions is to optimize the delivery vectors for RDOs. To date, few RDO delivery systems have been used. Therefore, new vectors should be tried for RDO transfer, such as the use of nanoparticles. Additionally, different kinds of modifications should be applied to RDO carrier systems to increase the total correction efficiency in vivo. Only with the development of delivery systems can RDOs be used for gene therapy, and successfully applied to functional genomics.     

Unmodified oligodeoxynucleotides require single-strandedness to induce targeted repair of a chromosomal EGFP gene

BACKGROUND: A number of genetic defects in humans are due to point mutations in a single, often tightly regulated gene. Genetic treatment of such defects is preferably done by correcting only the altered base pair at the endogenous locus rather than by a gene replacement strategy involving viral vectors. Promisingly high repair rates have been achieved in some systems with the non-viral approach of transfecting chimeric RNA/DNA oligonucleotides (chimeraplasts). However, since this technique does not yet perform robustly, several parameters thought to be important in oligonucleotide-mediated gene repair were examined. METHODS: A series of transgenic HEK-293 cell clones has been established harboring high or low copy numbers of a point-mutated 'enhanced green fluorescent protein' (EGFP) gene as the target. At the level of single living cells, repair efficiencies were measured by fluorescence-activated cell sorting (FACS) regarding topology (single-stranded, double-stranded), exonuclease protection (four phosphorothioate linkages at both ends), polarity (sense, antisense), and length (13mer, 19mer, 35mer, 69mer) of the oligonucleotide. RESULTS: When targeting chromosomal loci, up to 0.2% corrected cells were obtained with single-stranded unmodified oligodeoxynucleotides, whereas a chimeraplast, its DNA analogue, and double-stranded DNA fragments were practically non-functional. Correction efficiencies correlated with target gene copy numbers. Modifying exonuclease resistance, polarity or length of single-stranded oligodeoxynucleotides did not enhance repair efficacy above the sub-percentage range. CONCLUSIONS: Successful chromosomal reporter gene repair in HEK-293 cells required an oligodeoxynucleotide to be single-stranded. In concert with the gene copy number correlation, functional interaction between the repair molecule and the target site seems to be one bottleneck in targeted gene repair.     

Short,single-stranded oligonucleotides mediate targeted nucleotide conversion using extracts from isolated liver mitochondria

Site-specific single-nucleotide changes in chromosomal DNA of eukaryotic cells have been achieved using chimeric RNA/DNA oligonucleotides (ONs) and short single-stranded (SS) ONs. However, a variety of human diseases originate from single-point mutations in the genome of mitochondrial DNA. We previously demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate targeted single-nucleotide changes using chimeric ONs in vitro. However, different factor(s) and/or mechanism(s) appear to be involved in SS and RNA/DNA ON mediated DNA repair. Because mitochondria are deficient in certain factors involved in nuclear DNA repair pathways, we investigated whether mitochondria possess the enzymatic machinery for SS ON mediated DNA alterations. Using in vitro DNA repair assays based on mutagenized plasmids and a bacterial read-out system, SS ONs were designed to correct the point mutations in the genes encoded by the different plasmids. In this system, protein extracts from purified rat liver mitochondria and nuclei catalyzed similar levels of site-specific nucleotide modifications using SS ONs. Interestingly, extracts isolated from quiescent liver mediated significantly higher conversion rates than those isolated from regenerating liver. The results suggest that mitochondria contain the factors necessary for correction of single-point mutations by SS ONs. In addition, at least some are different than those required for DNA repair by RNA/DNA ONs. Moreover, correction with SS ONs appears to occur one strand at a time suggesting that repair of the DNA substrate involves strand transfer. The ability of unmodified SS ONs to mediate targeted alteration of the mitochondrial genome may provide a new tactic for treatment of certain mitochondrial-based diseases.     

Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides. Targeted delivery of liposomes and polyethyleneimine to the asialoglycoprotein receptor

Chimeric RNA/DNA oligonucleotides have been shown to promote single nucleotide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser365 position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and negatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stability and efficiency in targeting hepatocytes via the asialoglycoprotein receptor. Physical characterization and electron microscopy revealed that the oligonucleotides were efficiently encapsulated within the liposomes, with the positive and negative formulations remaining stable for at least 1 month. Transfection efficiencies in isolated rat hepatocytes approached 100% with each of the formulations. However, the negative liposomes and 25-kDa lactosylated polyethyleneimine provided the most intense nuclear fluorescence with the fluorescein-labeled oligonucleotides. The lactosylated polyethyleneimine and the three different liposomal formulations resulted in A to C conversion efficiencies of 19-24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatocytes. The results suggest that both the liposomal and polyethyleneimine formulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the introduction or repair of genetic mutations by the chimeric RNA/DNA oligonucleotides.     

Spontaneous mutation frequency in plants obscures the effect of chimeraplasty

In this study we tested the performance of chimeraplasts, chimeric RNA/DNA oligonucleotides, for the creation of directed changes in chromosomal sequences in tobacco and oilseed rape. As target genes for chimeraplasty, the endogenous als gene and two transgenes, bar and a fusion between egfp and bar, were used. In experiments in which similar numbers of cells were treated with and without chimeraplasts, delivery of chimeraplasts did not lead to increased numbers of herbicide-resistant or egfp fluorescent calli. Sequence analysis of the target genes revealed a range of sequence changes rather than the sequence modification intended through chimeraplast design. Importantly, in both the presence and absence of chimeraplasts, similar types of changes were obtained at similar frequencies. These data suggest that the observed changes at the target codon result primarily from spontaneous mutation.