DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation

Non-small cell lung cancer (NSCLC) patients treated with small molecule EGFR inhibitors, such as gefitinib, frequently develop drug resistance due to the presence of secondary mutations like the T790M mutation on EGFR exon 20. These mutations may originate from small subclonal populations in the primary tumor that become dominant later on during treatment. In order to detect these low-level DNA variations in the primary tumor or to monitor their progress in plasma, it is important to apply reliable and sensitive mutation detection methods. Here, we combine two recently developed methodologies, Differential Strand Separation at Critical Temperature (DISSECT), with peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) for the detection of T790M EGFR mutation. DISSECT pre-enriches low-abundance T790M EGFR mutations from target DNA prior to implementing PNA-LNA PCR, a method that can detect 1 mutant allele in a background of 100–1000 wild type alleles. The combination of DISSECT and PNA-LNA PCR enables the detection of 1 mutant allele in a background of 10,000 wild type alleles. The combined DISSECT-PNA-LNA PCR methodology is amenable to adaptation for the sensitive detection of additional emerging resistance mutations in cancer.     

Quantitation of the common components of deoxyribonucleic acids by mass spectrometry: application to the analysis of DNAs of unusual composition

A mass spectral method for the quantitation of the percentages of deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine in intact DNAs has been devised. Standard curves for each nucleoside have been constructed which are based upon the observation that a direct correlation exists between the heights (% deflection) of diagnostic peaks from these nucleosides in a mass spectrum and the published percent composition of specific DNAs. Analyses of DNA from Clostridiumperfringens, Micrococcusluteus, Escherichiacoli, Bacillussubtilis, Pseudomonasfluorescens, Drosophilamelanogaster, salmon sperm, and bacteriophage lambda were used to determine standard curves. The validity of the method was demonstrated by comparison of the results from the mass spectral procedure with results from the chemical analyses of the DNAs from calf thymus and wheat germ. Analysis of ØX-174 DNA yielded values consistent with the published values obtained via sequence analysis and indicated that the method is applicable to both single and double-stranded DNAs. Results from T2 DNA, which contains no cytidine, exhibited artificially high values for adenosine, guanosine and thymidine with concomitant alteration in the A/T and G/C molar ratios. Such skewed results are useful in predicting the presence of modified nucleosides. The extreme sensitivity of the method has been exploited in the analysis of subnanogram quantities of restriction endonuclease fragments from DNA.     

Allele-specific copy number profiling by next-generation DNA sequencing

The progression and clonal development of tumors often involve amplifications and deletions of genomic DNA. Estimation of allele-specific copy number, which quantifies the number of copies of each allele at each variant loci rather than the total number of chromosome copies, is an important step in the characterization of tumor genomes and the inference of their clonal history. We describe a new method, falcon, for finding somatic allele-specific copy number changes by next generation sequencing of tumors with matched normals. falcon is based on a change-point model on a bivariate mixed Binomial process, which explicitly models the copy numbers of the two chromosome haplotypes and corrects for local allele-specific coverage biases. By using the Binomial distribution rather than a normal approximation, falcon more effectively pools evidence from sites with low coverage. A modified Bayesian information criterion is used to guide model selection for determining the number of copy number events. Falcon is evaluated on in silico spike-in data and applied to the analysis of a pre-malignant colon tumor sample and late-stage colorectal adenocarcinoma from the same individual. The allele-specific copy number estimates obtained by falcon allows us to draw detailed conclusions regarding the clonal history of the individual''s colon cancer.     

Native Variants of the MRB1 Complex Exhibit Specialized Functions in Kinetoplastid RNA Editing

Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3’ to 5’ direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a ‘core’ complex of proteins but differ in the composition of the ‘variable’ proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.     

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.     

The human telomeric proteome during telomere replication

Telomere shortening can cause detrimental diseases and contribute to aging. It occurs due to the end replication problem in cells lacking telomerase. Furthermore, recent studies revealed that telomere shortening can be attributed to difficulties of the semi-conservative DNA replication machinery to replicate the bulk of telomeric DNA repeats. To investigate telomere replication in a comprehensive manner, we develop QTIP-iPOND - Quantitative Telomeric chromatin Isolation Protocol followed by isolation of Proteins On Nascent DNA - which enables purification of proteins that associate with telomeres specifically during replication. In addition to the core replisome, we identify a large number of proteins that specifically associate with telomere replication forks. Depletion of several of these proteins induces telomere fragility validating their importance for telomere replication. We also find that at telomere replication forks the single strand telomere binding protein POT1 is depleted, whereas histone H1 is enriched. Our work reveals the dynamic changes of the telomeric proteome during replication, providing a valuable resource of telomere replication proteins. To our knowledge, this is the first study that examines the replisome at a specific region of the genome.     

H-NS Can Facilitate Specific DNA-binding by RNA Polymerase in AT-rich Gene Regulatory Regions

Extremely AT-rich DNA sequences present a challenging template for specific recognition by RNA polymerase. In bacteria, this is because the promoter −10 hexamer, the major DNA element recognised by RNA polymerase, is itself AT-rich. We show that Histone-like Nucleoid Structuring (H-NS) protein can facilitate correct recognition of a promoter by RNA polymerase in AT-rich gene regulatory regions. Thus, at the Escherichia coli ehxCABD operon, RNA polymerase is unable to distinguish between the promoter −10 element and similar overlapping sequences. This problem is resolved in native nucleoprotein because the overlapping sequences are masked by H-NS. Our work provides mechanistic insight into nucleoprotein structure and its effect on protein-DNA interactions in prokaryotic cells.     

The molecular complexity of mu and pi symbiont DNA of Paramecium aurelia

The molecular size of mu and pi symbionts of Parameciumaurelia has been calculated from renaturation kinetic data. Observed values were 0.78 × 10⁹ daltons for mu particle DNA and 0.81 × 10⁹ daltons for pi particle DNA. Estimates of analytical complexity were 4.45 × 10⁹ and 5.05 × 10⁹ daltons respectively. Based on these data, mu and pi symbionts appear to possess multiple genomes and contain a minimum of 5 or 6 copies of each DNA sequence.     

Structure/Function Analysis of PARP-1 in Oxidative and Nitrosative Stress-Induced Monomeric ADPR Formation

Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2^nd messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1′s structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1′s BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2^nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.     

Evolution of the B3 DNA Binding Superfamily: New Insights into REM Family Gene Diversification

Background

The B3 DNA binding domain includes five families: auxin response factor (ARF), abscisic acid-insensitive3 (ABI3), high level expression of sugar inducible (HSI), related to ABI3/VP1 (RAV) and reproductive meristem (REM). The release of the complete genomes of the angiosperm eudicots Arabidopsis thaliana and Populus trichocarpa, the monocot Orysa sativa, the bryophyte Physcomitrella patens,the green algae Chlamydomonas reinhardtii and Volvox carteri and the red algae Cyanidioschyzon melorae provided an exceptional opportunity to study the evolution of this superfamily.

Methodology

In order to better understand the origin and the diversification of B3 domains in plants, we combined comparative phylogenetic analysis with exon/intron structure and duplication events. In addition, we investigated the conservation and divergence of the B3 domain during the origin and evolution of each family.

Conclusions

Our data indicate that showed that the B3 containing genes have undergone extensive duplication events, and that the REM family B3 domain has a highly diverged DNA binding. Our results also indicate that the founding member of the B3 gene family is likely to be similar to the ABI3/HSI genes found in C. reinhardtii and V. carteri. Among the B3 families, ABI3, HSI, RAV and ARF are most structurally conserved, whereas the REM family has experienced a rapid divergence. These results are discussed in light of their functional and evolutionary roles in plant development.     

Boosting riboswitch efficiency by RNA amplification

Riboswitches are RNA sensors that regulate gene expression in response to binding of small molecules. Although they conceptually represent simple on/off switches and, therefore, hold great promise for biotechnology and future synthetic biology applications, the induction of gene expression by natural riboswitches after ligand addition or removal is often only moderate and, consequently, the achievable expression levels are not very high. Here, we have designed an RNA amplification-based system that strongly improves the efficiency of riboswitches. We have successfully implemented the method in a biological system for which currently no efficient endogenous tools for inducible (trans)gene expression are available: the chloroplasts of higher plants. We further show that an HIV antigen whose constitutive expression from the chloroplast genome is deleterious to the plant can be inducibly expressed under the control of the RNA amplification-enhanced riboswitch (RAmpER) without causing a mutant phenotype, demonstrating the potential of the method for the production of proteins and metabolites that are toxic to the host cell.     

A Multiple Aminoacyl-tRNA Synthetase Complex That Enhances tRNA-Aminoacylation in African Trypanosomes

The genes for all cytoplasmic and potentially all mitochondrial aminoacyl-tRNA synthetases (aaRSs) were identified, and all those tested by RNA interference were found to be essential for the growth of Trypanosoma brucei. Some of these enzymes were localized to the cytoplasm or mitochondrion, but most were dually localized to both cellular compartments. Cytoplasmic T. brucei aaRSs were organized in a multiprotein complex in both bloodstream and procyclic forms. The multiple aminoacyl-tRNA synthetase (MARS) complex contained at least six aaRS enzymes and three additional non-aaRS proteins. Steady-state kinetic studies showed that association in the MARS complex enhances tRNA-aminoacylation efficiency, which is in part dependent on a MARS complex-associated protein (MCP), named MCP2, that binds tRNAs and increases their aminoacylation by the complex. Conditional repression of MCP2 in T. brucei bloodstream forms resulted in reduced parasite growth and infectivity in mice. Thus, association in a MARS complex enhances tRNA-aminoacylation and contributes to parasite fitness. The MARS complex may be part of a cellular regulatory system and a target for drug development.