Base Pair Positions Research Articles

Simulation of Radiation-Induced DNA Damage and Protection by Histones Using the Code RITRACKS.

(1) Background: DNA damage is of great importance in the understanding of the effects of ionizing radiation. Various types of DNA damage can result from exposure to ionizing radiation, with clustered types considered the most important for radiobiological effects. (2) Methods: The code RITRACKS (Relativistic Ion Tracks), a program that simulates stochastic radiation track structures, was used to simulate DNA damage by photons and ions spanning a broad range of linear energy transfer (LET) values. To perform these simulations, the transport code was modified to include cross sections for the interactions of ions or electrons with DNA and amino acids for ionizations, dissociative electron attachment, and elastic collisions. The radiochemistry simulations were performed using a step-by-step algorithm that follows the evolution of all particles in time, including reactions between radicals and DNA structures and amino acids. Furthermore, detailed DNA damage events, such as base pair positions, DNA fragment lengths, and fragment yields, were recorded. (3) Results: We report simulation results using photons and the ions 1H+, 4He2+, 12C6+, 16O8+, and 56Fe26+ at various energies, covering LET values from 0.3 to 164 keV/µm, and performed a comparison with other codes and experimental results. The results show evidence of DNA protection from damage at its points of contacts with histone proteins. (4) Conclusions: RITRACKS can provide a framework for studying DNA damage from a variety of ionizing radiation sources with detailed representations of DNA at the atomic scale, DNA-associated proteins, and resulting DNA damage events and statistics, enabling a broader range of future comparisons with experiments such as those based on DNA sequencing.

Identification of orange color-related gene, PhcpcC, in Pyropia haitanensis

Pigmentation-related mutations can be utilized to distinguish between differentially colored sectors of chimeric thalli, thereby facilitating the efficient breeding of economically valuable Pyropia/Porphyra seaweed species. However, the specific trait loci and alleles responsible for Pyropia/Porphyra coloration have yet to be identified, which limits the applicability of coloration mutants for breeding and genetic analyses. In this study, to preserve the genetic integrity of the population, only four-colored thalli were considered when constructing the doubled haploid (DH) Pyropia haitanensis population, which consisted of 480 homozygous offspring lines (representing the largest DH Pyropia/Porphyra population). The offspring lines in the DH population exhibited both wild-type colored and orange sectors, with a segregation ratio of approximately 1:1, indicating that the orange coloration was controlled by a single nuclear gene. Through BSA-seq analysis (99% confidence interval), a candidate region of 0.5 Mb was identified in the P. haitanensis genome. Additionally, a non-synonymous SNP [A/G] was detected at base-pair position 481 in the coding region of PhcpcC, which encodes a phycocyanin-associated rod linker protein. This SNP locus was verified in both DH and natural populations, with the wild-type colored lines having an A base and the orange lines having a G base at this locus. Therefore, PhcpcC may be the gene associated with the orange coloration of P. haitanensis. The molecular marker developed in this study can be employed to exploit pigmentation mutants for breeding and genetic analyses of Pyropia/Porphyra species.

Farnesoid X receptor represses human sulfotransferase 1A3 expression through direct binding to the promoter.

Human sulfotransferases 1A3 (SULT1A3) has received particular interest, due to their functions of catalyzing the sulfonation of numerous phenolic substrates, including bioactive endogenous molecules and therapeutic agents. However, the regulation of SULT1A3 expression and the underlying mechanism remain unclear. Here, we aimed to investigate the regulation effects of bile acid-activated farnesoid X receptor (FXR) on SULT1A3 expression, and to shed light on the mechanism thereof. Our results demonstrated that FXR agonists (CDCA and GW4064) significantly inhibit the expression of SULT1A3 at mRNA and protein levels. In addition, overexpression of FXR led to decrease in SULT1A3 expression and knockdown of FXR significantly induced the expression of SULT1A3 in protein and mRNA levels, confirming that FXR expression manifestly showed negative regulatory effect on basal SULT1A3 expression. Furthermore, a combination of luciferase reporter gene and CHIP assays showed that FXR repressed SULT1A3 transcription through direct binding to the region at base pair positions -664 to -654. In conclusion, this study for the first time confirmed FXR was a negative transcriptional regulator of human SULT1A3 enzyme.

Deciphering correlation and causation in risk factors for heart disease with Mendelian randomization

In this analysis, we studied the use of Mendelian randomization to identify the risk factors of coronary artery disease (CAD), a major cause of cardiovascular disease. Identifying risk factors of CAD are critical to understanding and managing the disease. Our analysis combined results from 28 genetic analyses from 12 unique studies. For each genetic variation, we obtained the variant ID, chromosome, base-pair position, reference and alternative alleles of the genetic variation, and estimated effect and p-value of the genetic variation on the outcome. We hypothesized that traits which are correlated with CAD outcomes will be causally associated with CAD risk in a genetic Mendelian randomization analysis. Our analysis showed that several traits such as blood pressure readings (systolic, OR 0.51 (95% CI: 0.34-0.69), p-value = 5.4x10-9) and (diastolic, OR 0.56 (95% CI: 0.41-0.71), p-value = 7.6x10-14), LDL cholesterol levels (OR 0.54 (95% CI: 0.47-0.60), p-value = 4.4x10-56), and BMI (OR 0.41 (95% I: 0.35-0.48), p-value = 6.30x10-33) were significant risk factors for CAD. C-reactive protein (OR -0.09 (95% CI: -0.18–0.00), p-value = 0.05) was a protective risk factor of CAD due to its negative odds ratio. In contrast, eosinophil count (OR -0.007 (95% CI: -0.06-0.04), p-value = 0.79) had no statistically significant association. Blood cells had weak associations with CAD, and uric acid’s role as a causal or reversible risk factor of CAD was inconclusive, requiring further study.

STRling: a k-mer counting approach that detects short tandem repeat expansions at known and novel loci

Expansions of short tandem repeats (STRs) cause many rare diseases. Expansion detection is challenging with short-read DNA sequencing data since supporting reads are often mapped incorrectly. Detection is particularly difficult for “novel” STRs, which include new motifs at known loci or STRs absent from the reference genome. We developed STRling to efficiently count k-mers to recover informative reads and call expansions at known and novel STR loci. STRling is sensitive to known STR disease loci, has a low false discovery rate, and resolves novel STR expansions to base-pair position accuracy. It is fast, scalable, open-source, and available at: github.com/quinlan-lab/STRling.

SumStatsRehab: an efficient algorithm for GWAS summary statistics assessment and restoration

BackgroundGenerating polygenic risk scores for diseases and complex traits requires high quality GWAS summary statistic files. Often, these files can be difficult to acquire either as a result of unshared or incomplete data. To date, bioinformatics tools which focus on restoring missing columns containing identification and association data are limited, which has the potential to increase the number of usable GWAS summary statistics files.ResultsSumStatsRehab was able to restore rsID, effect/other alleles, chromosome, base pair position, effect allele frequencies, beta, standard error, and p-values to a better extent than any other currently available tool, with minimal loss.ConclusionsSumStatsRehab offers a unique tool utilizing both functional programming and pipeline-like architecture, allowing users to generate accurate data restorations for incomplete summary statistics files. This in turn, increases the number of usable GWAS summary statistics files, which may be invaluable for less researched health traits.

P-OGC39 The smell of oesophageal adenocarcinoma: opportunities for tests and treatments

Abstract Background Exhaled breath analysis is a promising approach for oesophageal adenocarcinoma (OAC) early detection. The biomarkers of interest are low molecular weight metabolites including volatile aldehydes. In this translational study we investigated whether these metabolites originated from a tumoral source, and how this might impact the diagnosis and treatment of OAC patients. Methods The investigative strategy was directed by an unbiased informatics screen of metabolic reprogramming in OAC, and validated using complimentary gene expression assays (n = 638, including controls). Mass spectrometric methods were used to quantify corresponding metabolites and putative source compounds at a tissue level (n = 158), and also in exhaled breath for correlative purposes. Targeted in vitro experiments were performed to demonstrate the cause and effect of the proposed model of metabolic reprogramming in OAC. Results The unbiased screen and subsequent validation found that reduced aldehyde detoxification is an OAC hallmark. In vitro and in vivo this was associated with endogenous aldehyde accumulation. OAC tissue was generally enriched for volatile aldehydes, including the genotoxins formaldehyde, acetaldehyde, 4-hydroxy-2-nonenal and 2-butenal, and the exhaled biomarker decanal (all P < 0.0001). Decanal concentrations correlated with exhaled concentrations. Considering potential aldehyde sources, the OAC phospholipidome was characterised by desaturated and longer lipid acyls, and these spontaneously generated biomarker aldehyde species at ambient conditions. Enriched genotoxic aldehydes were detectable in base-pairing positions in DNA; this genotoxicity was therapeutically targetable with aldehyde scavengers in vitro. Conclusions These data support a model for enriched exhaled aldehydes based on increased production from an altered lipid phenotype, and reduced detoxification. Some aldehydes are non-reactive and thus support non-invasive detection. Others react with DNA and increase local genotoxicity; this process is druggable. These findings have implications for OAC early diagnosis and chemoprevention.

Characterization of G4 DNA formation in mitochondrial DNA and their potential role in mitochondrial genome instability.

Mitochondria possess their own genome which can be replicated independently of nuclear DNA. Mitochondria being the powerhouse of the cell produce reactive oxygen species, due to which the mitochondrial genome is frequently exposed to oxidative damage. Previous studies have demonstrated an association of mitochondrial deletions to aging and human disorders. Many of these deletions were present adjacent to non-B DNA structures. Thus, we investigate noncanonical structures associated with instability in mitochondrial genome. Insilico studies revealed the presence of >100 G-quadruplex motifs (of which 5 have the potential to form 3-plate G4 DNA), 23 inverted repeats, and 3 mirror repeats in the mitochondrial DNA (mtDNA). Further analysis revealed that among the deletion breakpoints from patients with mitochondrial disorders, majority are located at G4 DNA motifs. Interestingly, ~50% of the deletions were at base-pair positions 8271-8281, ~35% were due to deletion at 12362-12384, and ~12% due to deletion at 15516-15545. Formation of 3-plate G-quadruplex DNA structures at mitochondrial fragile regions was characterized using electromobility shift assay, circular dichroism (CD), and Taq polymerase stop assay. All 5 regions could fold into both intramolecular and intermolecular G-quadruplex structures in a KCl-dependent manner. G4 DNA formation was in parallel orientation, which was abolished in the presence of LiCl. The formation of G4 DNA affected both replication and transcription. Finally, immunolocalization of BG4 with MitoTracker confirmed the formation of G-quadruplex in mitochondrial genome. Thus, we characterize the formation of 5 different G-quadruplex structures in human mitochondrial region, which may contribute toward formation of mitochondrial deletions.

A quantum mechanical approach to random X chromosome inactivation

<abstract> <p>The X chromosome inactivation is an essential mechanism in mammals' development, that despite having been investigated for 60 years, many questions about its choice process have yet to be fully answered. Therefore, a theoretical model was proposed here for the first time in an attempt to explain this puzzling phenomenon through a quantum mechanical approach. Based on previous data, this work theoretically demonstrates how a shared delocalized proton at a key base pair position could explain the random, instantaneous, and mutually exclusive nature of the choice process in X chromosome inactivation. The main purpose of this work is to contribute to a comprehensive understanding of the X inactivation mechanism with a model proposal that can complement the existent ones, along with introducing a quantum mechanical approach that could be applied to other cell differentiation mechanisms.</p> </abstract>

Differential gene expression in dairy cows under negative energy balance and ketosis: A systematic review and meta-analysis

Development of ketosis in high-producing dairy cows contributes to several animal health issues and highlights the need for a better understanding of the genetic basis of metabolic diseases. To evaluate the pattern of differential gene expression in the liver of cows under negative energy balance (NEB), and under subclinical and clinical ketosis, a meta-analysis of gene expression and genome-wide association studies results was performed. An initial systematic review identified 118 articles based on the key words "cow," "liver," "negative energy balance," "ketosis," "expression," "qPCR," "microarray," "proteomic," "RNA-Seq," and "GWAS." After further screening for only peer-reviewed and pertinent articles for gene expression during NEB and clinical and subclinical ketosis (considering plasma levels of β-hydroxybutyrate), 20 articles were included in the analysis. From the systematic review, 430 significant SNPs identified by genome-wide association studies (GWAS) were assigned to genes reported in gene expression studies by considering chromosome and base pair positions in the ARS-UCD 1.2 bovine assembly. Venn diagrams were created to integrate the data obtained in the systematic review, and Gene Ontology enrichment analysis was carried out using official gene names. A QTL enrichment analysis was also performed to identify potential positional candidate loci. Twenty-four significant SNPs were located within the coordinates of differentially expressed genes located on chromosomes 2, 3, 6, 9, 11, 14, 27, and 29. Three significant metabolic pathways were associated with NEB and subclinical and clinical ketosis. In addition, 2 important genes, PPARA (peroxisome proliferator activated receptor alpha) and ACACA (acetyl-coenzyme A carboxylase α), were identified, which were differentially expressed in the 3 metabolic conditions. The PPARA gene is involved in the regulation of lipid metabolism and fatty liver disease and the ACACA gene encodes an enzyme that catalyzes the carboxylation of acetyl-coenzyme A to malonyl-coenzyme A, which is a rate-limiting step in fatty acid synthesis. Gene network analysis revealed co-expression interactions among 34 genes associated with functions involving fatty acid transport and fatty acid metabolism. For the annotated QTL, 9 QTL were identified for ketosis. The genes FN1 (fibronectin 1) and PTK2 (protein tyrosine kinase 2), which are mainly involved in cell adhesion and formation of extracellular matrix constituents, were enriched for QTL previously associated with the trait "ketosis" on chromosome 2 and for the trait "milk iron content" on chromosome 14, respectively. This integration of gene expression and GWAS data provides an additional understanding of the genetic background of NEB and subclinical and clinical ketosis in dairy cattle. Thus, it is a useful approach to identify biological mechanisms underlying these metabolic conditions in dairy cattle.

Genetic diversity analysis of Corynespora cassiicola isolates on the rubber tree (Hevea brasiliensis) in Vietnam using ribosomal DNA internal transcribed spacer (rDNA-ITS) sequences and sequence-related amplified polymorphism (SRAP)

A total of 76 Corynespora cassiicola isolates collected from 16 rubber clones in several geographic regions of Vietnam, were studied for genetic diversity by ribosomal DNA internal transcribed spacer (rDNA-ITS) sequences and sequence-related amplified polymorphism (SRAP) techniques. Results of rDNA-ITS sequence analysis indicated 2 nucleotide polymorphisms at the 135th (cytosine/thymine) and 474th (guanine/adenine) base pair positions of rDNA-ITS region, which differentiated the 76 studied isolates into 3 clusters. All isolates could be uniquely distinguished by 208 polymorphism bands which generated 93.3% polymorphic ratio with SRAP markers. A dendrogram produced from UPGMA analysis based on Nei and Li’s coefficient divided the 76 C. cassiicola isolates into 2 main clusters. Cluster one included 54 fungal isolates of which 51 and 3 were observed in subgroup 1A and 1B, respectively. There were 22 C. cassiicola isolates belonging to cluster 2 with subgroups 2A and 2B consisted of 20 and 2 fungal isolates, respectively. Bootstrap values for groups one and two were 61% and 100%, respectively and the grouping was reliable, with similarity coefficient between the two main groups at 67%. rDNA-ITS sequence analysis revealed a new genetic group, signifying at least three C. cassiicola genetic groups that infect rubber trees in Vietnam. SRAP markers divided the studied isolates into two distinct groups, which correlated with geographical location rather than host source. To the best of our knowledge, this is the first demonstration using SRAP markers on the systematics of C. cassiicola isolates which cause the Corynespora leaf fall disease on various rubber (Hevea brasiliensis) clones in Vietnam.

Genome-wide association study of birth weight and pre-weaning body weight of crossbred pigs

In piggery, birth weight and body weight remains most vital economic trait as they directly influence on the production performance of the farm. Implementing the genomic selection would pay way for rapid genetic gain along with increased accuracy than conventional breeding. Prior to genomic selection, genome wide association study (GWAS) has to be conducted in order to find informative SNPs associated with the traits of interest in a given population. Under this study 96 crossbred pigs were genotyped using double digest genotype by sequencing (GBS) technique using Hiseq platform. Raw FASTQ data were processed using dDOCENT Pipeline on Reference based method and variants were called using Free Bayes (version 1.1.0-3). Using Plink (v1.09b), variants having MAF>0.01, HWE<0.001 and genotyping rate >80% were filtered out and 20,467 SNPs were retained after quality control, for ascertaining GWAS in 96 pigs. Before conducting association studies, the data were adjusted for significant nongenetic factors affecting the traits of interest. GWAS was performed using Plink software (v1.9b) identified 9, 11, 12, 23, 28, 24, 30, 33 and 42 SNPs significantly (adjusted P<0.001) associated with birth weight, body weight at weekly interval from 1st week to 8th week, respectively. A large proportion of significant (adjusted P<0.001) SNPs were located on SSC10, SSC6, SSC13, SSC8 and SSC1. One genome wide significant SNP and four genome wide suggestive SNPs were identified. Two common SNPs affecting all body weight at different weeks were located on SSC5:40197442 and SSC13:140562 base pair position. This study helps to identify the genome wide scattered significant SNPs associated with traits of interest which could be used for genomic selection, but further validation studies of these loci in larger population are recommended.

Characterization of an Expressed Triticum monococcum Glu-A1y Gene Containing a Premature Termination Codon in its C-terminal Coding Region

Premature termination codons (PTCs) are an important reason for the silence of high-molecular-weight glutenin subunits in Triticum species. Although the Glu-A1y gene is generally silent in common wheat, we here isolated an expressed Glu-A1y gene containing a PTC, named 1Ay8.3, from Triticum monococcum ssp. monococcum (AmAm, 2n = 2x = 14). Despite the presence of a PTC (TAG) at base pair positions 1879–1881 in the C-terminal coding region, this did not obviously affect 1Ay8.3 expression in seeds. This was demonstrated by the fact that when the PTC TAG of 1Ay8.3 was mutated to the CAG codon, the mutant in Escherichia coli bacterial cells expressed the same subunit as in the seeds. However, in E. coli, 1Ay8.3 containing the PTC expressed a truncated protein with faster electrophoretic mobility than that in seeds, suggesting that PTC translation termination suppression probably occurs in vivo (seeds) but not in vitro (E. coli). This may represent one of only a few reports on the PTC termination suppression phenomenon in genes.

Molecular characterization of a novel Muscovy duck parvovirus isolate: evidence of recombination between classical MDPV and goose parvovirus strains

BackgroundMuscovy duck parvovirus (MDPV) and Goose parvovirus (GPV) are important etiological agents for Muscovy duck parvoviral disease and Derzsy’s disease, respectively; both of which can cause substantial economic losses in waterfowl industry. In contrast to GPV, the complete genomic sequence data of MDPV isolates are still limited and their phylogenetic relationships largely remain unknown. In this study, the entire genome of a pathogenic MDPV strain ZW, which was isolated from a deceased Muscovy duckling in 2006 in China, was cloned, sequenced, and compared with that of other classical MDPV and GPV strains.ResultsThe genome of strain ZW comprises of 5071 nucleotides; this genome was shorter than that of the pathogenic MDPV strain YY (5075 nt). All the four deleted nucleotides produced in strain ZW are located at the base-pairing positions in the palindromic stem of inverted terminal repeats (ITR) without influencing the formation of a hairpin structure. Recombination analysis revealed that strain ZW originated from genetic recombination between the classical MDPV and GPV strain. The YY strain of MDPV acts as the major parent, whereas the virulent strains YZ99–6 and B and the vaccine strain SYG61v of GPV act as the minor parents in varying degrees. Two recombination sites were detected in strain ZW, with the small recombination site surrounding the P9 promoter, and the large recombination site situated in the middle of the VP3 gene. The SYG61V strain is a vaccine strain used for preventing goose parvoviral disease. This strain was found to be solely involved in the recombination event detected in the P9 promoter region. Phylogenetic analyses between strain ZW and other classical strains of MDPV and GPV were performed. The results supported the in silico recombination analysis conclusion.ConclusionsMDPV Strain ZW is a novel recombinant parvovirus, and the bulk of its genome originates from the classical MDPV strain. Two virulent strains and a vaccine strain of GPV were involved in the recombination process in varying degrees.

English

The phylogenetic utility of the 16S gene in cichlids is assessed. Eighty-six (86) partial sequences belonging to 37 genera of cichlids from the Genbank was analyzed. The alignment had four hundred and sixty three (463) basepairs with 337 conserved sites and 126 variable sites. Base compositional bias is similar to that found in higher organism with Adenine having the highest average of 30.3%, followed by cytosine, guanine and thiamine with the average values of 26.1, 21.9 and 21.7% respectively. The most suitable evolutionary model is the K2+G+I model as this had the lowest Bayesian Information Criterion. There were 4 major indels at basepair positions 328 which is unique to the Heterotilapia buttikoferi, position 369 unique to Gramatoria lemarii, position 396 which is shared by Tilapia sparrmanii, T. guinasana and T. zilli. The indel at position 373 was found in all tested species except the Oreochromis mossambicus. The Tilapine general is the basal group in Cichlids. The 16S gene separates the Tilapia genera without any ambiguity but there were phylogenetic overlaps in the Sarotherodon and Oreochromis. More finite molecular and statistical methodology may be needed to distinguish the Sarotherodon and Oreochromis. The diversity of cichlids is generally very low due to a common ancestry with little differentiation genetically. The grouping of the Oreochromis and Sarotherodon genera together in the same clade is not unconnected with the preservation of genetic beacons that the group retained as it evolved. Key words: Cichlids, 16S gene, phylogeny, evolutionary model, monophyletic, conserved segment, speciose, indels

Abstract LB-167: Off-target analysis of RNAi screens identifies microRNA seed sequence contributing to metastatic invasion

Abstract The occurrence of metastatic events is a prominent indicator of poor survival outcomes in individuals afflicted by solid tumor cancers. The introduction of high throughput techniques for genetic knockdown or ablation, such as shRNA and CRISPR-Cas9 libraries, provides the means to effectively screen for loss of function genes that contribute to the metastatic cascade; however, screening strategies employing these methods, particularly RNAi, carry an inherent risk of false positives due to off-target effects. These off-target effects are often mediated by shRNA sequences mimicking microRNA functionality, whereby only seed sequence (base pair positions 2-8) complementarity to the 3’ UTR of transcripts is sufficient for effective targeting and downregulation. Through two separate genome-wide shRNA screens, we identified a particular shRNA that shares its seed sequence with an uncharacterized miR, miR-8087, and exhibits wide-spread, miR-like off-target effects causing increased chemotaxis and Matrigel invasiveness. A comprehensive, third generation shRNA library was utilized to screen for metastasis suppressor genes in non-aggressive breast and prostate cancer cell lines (T-47D and LNCaP; respectively) by both in vitro and in vivo methods. Enriched shRNA variants were identified by barcode sequencing of clones and one shRNA sequence in particular, targeting apolipoprotein L-II (APOL2), was consistently identified in both screens. Conversion of the shAPOL2 sequence to siRNA and subsequent transfection in LNCaP cells increased the invasive and chemotactic potential of these cells. In contrast, APOL2 knockdown with alternative siRNA sequences did not increase the invasive or migratory capability of LNCaP cells. In order to identify off-targets responsible for the invasive phenotype, the gene expression profile of LNCaP transfected with the functionally relevant siAPOL2 was compared by microarray analysis (Illumina HT-12v4) to that of cells containing APOL2-specific siRNA. The library-specific siAPOL2 results in differential downregulation of 132 gene transcripts, suggesting miR-like activity. Examination of the siRNA seed sequence reveals exact homology to miR-8087 seed sequence and many of the siAPOL2 off-targets are shared with miR-8087 gene targets as predicted by miRWalk 2.0. Currently, efforts are underway to characterize miR-8087 expression and targeting in a panel of cell lines with varying migratory potentials as well as in patient primary and metastatic prostate cancer tissue samples. The off-target analysis of RNAi screens reveals a broad acting miR sequence that contributes to the invasive potential of cells and may provide a predictive and targetable element in metastatic cancers. Citation Format: Henry G. Withers, Ryan Ransom, Irwin H. Gelman. Off-target analysis of RNAi screens identifies microRNA seed sequence contributing to metastatic invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-167.

The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase

Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, Tm, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (Tm(F/T) < Tm(εA/T) < Tm(Hx/T) < Tm(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme–substrate complex is not the bottleneck controlling the catalytic activity of AAG.

Noncanonical registers and base pairs in human 5' splice-site selection.

Accurate recognition of splice sites is essential for pre-messenger RNA splicing. Mammalian 5′ splice sites are mainly recognized by canonical base-pairing to the 5′ end of U1 small nuclear RNA, yet we described multiple noncanonical base-pairing registers by shifting base-pair positions or allowing one-nucleotide bulges. By systematic mutational and suppressor U1 analyses, we prove three registers involving asymmetric loops and show that two-nucleotide bulges but not longer can form in this context. Importantly, we established that a noncanonical uridine-pseudouridine interaction in the 5′ splice site/U1 helix contributes to the recognition of certain 5′ splice sites. Thermal melting experiments support the formation of noncanonical registers and uridine-pseudouridine interactions. Overall, we experimentally validated or discarded the majority of predicted noncanonical registers, to derive a list of 5′ splice sites using such alternative mechanisms that is much different from the original. This study allows not only the mechanistic understanding of the recognition of a wide diversity of mammalian 5′ splice sites, but also the future development of better splice-site scoring methods that reliably predict the effects of disease-causing mutations at these sequences.

Cyst-theca relationship and phylogenetic positions of Scrippsiella plana sp. nov. and S. spinifera (Peridiniales, Dinophyceae)

Species belonging to the dinophyte genus Scrippsiella are frequently reported in marine waters, but information on their distribution in brackish environments is limited. Here we describe a new species, S. plana, through incubation of non-calcified cysts from sediments collected in the South China Sea and Caspian Sea. The vegetative cells consist of a conical epitheca and a rounded hypotheca with the plate formula of Po, X, 4′, 3a, 7′′, 5C+t, 5S, 5′′′, 2′′′′. It differs from other Scrippsiella species by its flattened body in dorsoventral view and a small first anterior intercalary (1a) plate (half the size of plate 3a). Scrippsiella plana strains from the South China Sea and Caspian Sea share identical internal transcribed spacer (ITS) sequences, and show phenotypic plasticity and local adaptation in growth rate at various salinities, consistent with the environments in which they originated. In addition, two strains of S. spinifera were obtained by incubating ellipsoid cysts with calcareous spines from sediments collected along the Turkish and Hawaiian coast. They also share identical ITS sequences and differ from Duboscquodinium collinii (a parasite of tintinnids) only at two base pair positions (in the ITS2 region). Molecular phylogeny based on ITS and large subunit ribosomal DNA (LSU rDNA) sequences revealed that S. plana was nested within the Calciodinellum (CAL) clade and S. spinifera within the S. trochoidea (STR) clade. The phylogenetic position of ‘Peridinium’ wisconsinense is reported for the first time, which supports multiple transitions of the Peridiniales to freshwater.

Optimal codons in Tremella fuciformis end in C/G, a strong difference with known Tremella species.

Tremella fuciformis is a popular edible fungus with fruiting bodies that can be produced in large quantities at low costs, while it is easy to transform and cultivate as yeast. This makes it an attractive potential bioreactor. Enhanced heterologous gene expression through codon optimization would be useful, but until now codon usage preferences in T. fuciformis remain unknown. To precisely determine the preferred codon usage of T. fuciformis we sequenced the genome of strain Tr26 resulting in a 24.2 Mb draft genome with 10,040 predicted genes. 3288 of the derived predicted proteins matched the UniProtKB/Swiss-Prot databases with 40% or more similarity. Corresponding gene models of this subset were subsequently optimized through repetitive comparison of alternative start codons and selection of best length matching gene models. For experimental confirmation of gene models, 96 random clones from an existing T. fuciformis cDNA library were sequenced, generating 80 complete CDSs. Calculated optimal codons for the 3288 predicted and the 80 cloned CDSs were highly similar, indicating sufficient accuracy of predicted gene models for codon usage analysis. T. fuciformis showed a strong preference for C and then G at the third base pair position of used codons, while average GC content of predicted genes was slightly higher than the total genome sequence average. Most optimal codons ended in C or G except for one, and an increased frequency of C ending codons was observed in genes with higher expression levels. Surprisingly, the preferred codon usage in T. fuciformis strongly differed from T. mesenterica and C. neoformans. Instead, optimal codon usage was similar to more distant related species such as Ustilago maydis and Neurospora crassa. Despite much higher overall sequence homology between T. fuciformis and T. mesenterica, only 7 out of 21 optimal codons were equal, whereas T. fuciformis shared up to 20 out of 21 optimal codons with other species. Clearly, codon usage in Tremella can differ largely and should be estimated for individual species. The precise identification of optimal and high expression related codons is therefore an important step in the development of T. fuciformis as a bioreactor system.