High Antioxidant Ability Confer Resistance to Atrazine in Commelina communis L.

Asiatic dayflower (Commelina communis L.) is a detrimental weed that mainly infests corn and soybean fields in China. Recently, some C. communis populations have exhibited resistance to atrazine, intensifying the difficulties in controlling the weed. However, little is known on the mechanism underlying C. communis resistance to atrazine. Therefore, two populations collected from Jilin (JL-1) and Jiangsu (JS-10) provinces of China were used to evaluate their growth responses to atrazine. The results showed that the JL-1 population displayed a low level of resistance to atrazine compared with JS-10 population, with the resistant index (RI) value of 2.9. To determine if a mutation in the psbA gene was the basis for varied resistance to this herbicide, the full-length gene encoding 353 amino acids with no intron was sequenced by using genome-walking techniques. No mutation known to confer resistance to atrazine was observed in either JL-1 or JS-10 populations. The malondialdehyde (MDA) contents relative to the control group were significantly higher in JS-10 population than in JL-1 population at 7 days after treatment with atrazine, suggesting that atrazine induced severer oxidant damage on JS-10 population. Additionally, significantly enhanced activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX), were detected in the JL-1 population, which was most likely to confer resistance to atrazine. To the best of our knowledge, this is the first investigation into the potential genetic and enzymatic differences contributing to atrazine resistance in this population.

Splicing Outcomes of 5′ Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays

Combining data derived from a meta-analysis of human disease-associated 5′ splice site GT>GC (i.e., +2T>C) variants and a cell culture-based full-length gene splicing assay (FLGSA) of forward engineered +2T>C substitutions, we recently estimated that ∼15–18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts. Herein, we analyzed the splicing outcomes of 20 +2T>C variants that generate some wild-type transcripts in two minigene assays. We found a high discordance rate in terms of the generation of wild-type transcripts, not only between FLGSA and the minigene assays but also between the different minigene assays. In the pET01 context, all 20 wild-type minigene constructs generated the expected wild-type transcripts; of the 20 corresponding variant minigene constructs, 14 (70%) generated wild-type transcripts. In the pSPL3 context, only 18 of the 20 wild-type minigene constructs generated the expected wild-type transcripts whereas 8 of the 18 (44%) corresponding variant minigene constructs generated wild-type transcripts. Thus, in the context of a particular type of variant, we raise awareness of the limitations of minigene splicing assays and emphasize the importance of sequence context in regulating splicing. Whether or not our findings apply to other types of splice-altering variant remains to be investigated.

Fox Hunting in Wild Apples: Searching for Novel Genes in Malus Sieversii

Malus sieversii is considered the progenitor of modern apple (Malus pumila) cultivars and to represent a valuable source of genetic diversity. Despite the importance of M. sieversii as a source of disease resistance, stress tolerance, and novel fruit traits, little is known about gene function and diversity in M. sieversii. Notably, a publicly annotated genome sequence for this species is not available. In the current study, the FOX (Full-length cDNA OvereXpressing) gene hunting system was used to construct a library of transgenic lines of Arabidopsis in which each transgenic line overexpresses a full-length gene obtained from a cDNA library of the PI619283 accession of M. sieversii. The cDNA library was constructed from mRNA obtained from bark tissues collected in late fall–early winter, a time at which many abiotic stress-adaptative genes are expressed. Over 4000 apple FOX Arabidopsis lines have been established from the pool of transgenic seeds and cDNA inserts corresponding to various Gene Ontology (GO) categories have been identified. A total of 160 inserts appear to be novel, with no or limited homology to M. pumila, Arabidopsis, or poplar. Over 1300 lines have also been screened for freezing resistance. The constructed library of transgenic lines provides a valuable genetic resource for exploring gene function and diversity in Malus sieversii. Notably, no such library of t-DNA lines currently exists for any Malus species.

Efficient Sequencing, Assembly, and Annotation of Human KIR Haplotypes

The homology, recombination, variation, and repetitive elements in the natural killer-cell immunoglobulin-like receptor (KIR) region has made full haplotype DNA interpretation impossible without physical separation of chromosomes. Here, we present a new approach using long-read sequencing to efficiently capture, sequence, and assemble diploid human KIR haplotypes. Sequences for capture probe design were derived from public full-length gene and haplotype sequences. IDT xGen® Lockdown probes were used to capture 2-8 kb of sheared DNA fragments followed by sequencing on a PacBio Sequel. The sequences were error corrected, binned, and then assembled using the Canu assembler. The assembly was evaluated on 16 individuals (8 African American and 8 Europeans) from whom ground truth was known via long-range sequencing on fosmid-isolated chromosomes. Using only 18 capture probes, the results show that the assemblies cover 97% of the GenBank reference, are 99.97% concordant, and it takes only 1.8 contigs to cover 75% of the reference. We also report the first assembly of diploid KIR haplotypes from long-read WGS, including the first sequencing of cB05∼tB01, which pairs a KIR2DS2/KIR2DS3 fusion with the tB01 region. Our targeted hybridization probe capture and sequencing approach is the first of its kind to fully sequence and phase all diploid human KIR haplotypes, and it is efficient enough for population-scale studies and clinical use.

Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth

Entamoeba histolytica is a globally important pathogen that is dramatically understudied. Its challenging genome has limited tractability. To enable forward genetics, we constructed the first genome-wide E. histolytica RNAi knockdown mutant library. The library is designed to enable deep sequencing analysis for quantitative identification of mutants after selection. We developed a novel analysis pipeline to precisely define and quantify full-length gene fragments inferred from read mapping. We performed the first E. histolytica RNAi screen and identified slow growth mutants. Growth phenotypes were reproducible in independently generated mutants. Some of the genes targeted in slow growth mutants had annotated functions consistent with roles in growth or metabolism. Some targeted genes lacked annotation, supporting the power of forward genetics in uncovering gene function. This work opens up the possibility of applying genetics to improve understanding of this important pathogen. Moreover, the strategies behind this RNAi library, and its analysis, are novel, and can be applied to other organisms.

RNA secondary structure mediated by Alu insertion as a novel disease-causing mechanism

ABSTRACTWe have recently reported a homozygous Alu insertion variant (termed Alu_Ins) within the 3’-untranslated region (3’-UTR) of the SPINK1 gene as the cause of a new pediatric disease entity. Although Alu-Ins has been shown, by means of a full-length gene expression assay (FLGEA), to result in the complete loss of SPINK1 mRNA expression, the precise underlying mechanism(s) has remained elusive. Herein, we filled this knowledge gap by adopting a hypothesis-driven approach. Employing RepeatMasker, we identified two Alu elements (termed Alu1 and Alu2) within the SPINK1 locus; both are located deep within intron 3 and, most importantly, reside in the opposite orientation to Alu-Ins. Using FLGEA, we provide convincing evidence that Alu-Ins disrupts splicing by forming RNA secondary structures with Alu1 in the pre-mRNA sequence. Our findings reveal a previously undescribed disease-causing mechanism, resulting from an Alu insertion variant, which has implications for Alu detection and interpretation in human disease genes.

Enterovirus 71 VP1 Protein Regulates Viral Replication in SH-SY5Y Cells via the mTOR Autophagy Signaling Pathway

Background: Enterovirus 71 (EV71) is the main pathogen that causes severe hand, foot, and mouth disease with fatal neurological complications. However, its neurovirulence mechanism is still unclear. Candidate virulence sites were screened out at structural protein VP1, but the function of these candidate virulence sites remains unclear. Several studies have shown that autophagy is associated with viral replication. However, the relationship between VP1 and autophagy in human neurons has not been studied. Methods: A recombinant virus—SDLY107-VP1, obtained by replacing the VP1 full-length gene of the SDLY107 strain with the VP1 full-length gene of the attenuated strain SDJN2015-01—was constructed and tested for replication and virulence. We then tested the effect of the recombinant virus on autophagy in nerve cells. The effect of autophagy on virus replication was detected by western blot and plaque test. Finally, the changes of mTOR signaling molecules during EV71 infection and the effect of mTOR on virus replication at the RNA level were detected. Results: Viral recombination triggered virulence attenuation. The replication ability of recombinant virus SDLY107-VP1 was significantly weaker than that of the parent strain SDLY107. The SDLY107 strain could inhibit autophagic flux and led to accumulation of autophagosomes, while the SDLY107-VP1 strain could not cause autophagosome accumulation. The synthesis of EV71 RNA was inhibited by inhibiting mTOR. Conclusions: Replacement of VP1 weakened the replication ability of virulent strains and reduced the level of autophagy in nerve cells. This autophagy facilitates the replication of virulent strains in nerve cells. VP1 is an important neurovirulence determinant of EV71, which affects virus replication by regulating cell autophagy. mTOR is a key molecule in this type of autophagy.

5’ splice site GC>GT variants differ from GT>GC variants in terms of their functionality and pathogenicity

ABSTRACTIn the human genome, most 5’ splice sites (~99%) employ the canonical GT dinucleotide whereas a small minority (~1%) use the non-canonical GC dinucleotide. The functionality and pathogenicity of 5’ splice site GT>GC (i.e., +2T>C) variants have been extensively studied but we still know very little about 5’ splice site GC>GT (+2C>T) variants. Herein, we sought to address this deficiency by performing a meta-analysis of identified +2C>T pathogenic variants together with a functional analysis of +2C>T substitutions using a cell culture-based full-length gene splicing assay. Our results establish a proof of concept that +2C>T variants are qualitatively different from +2T>C variants in terms of their functionality and pathogenicity and suggest that, in sharp contrast with +2T>C variants, most if not all +2C>T variants have no pathological relevance. Our findings have important implications for interpreting the clinical relevance of +2C>T variants but might also improve our understanding of the evolutionary basis of switching between GT and GC 5’ splice sites in mammalian genomes.

Analysis of the Full-Length Pyriform Spidroin Gene Sequence

Spiders often produce multiple types of silk, each with unique properties suiting them to certain tasks and biological functions. Orb-weaver spiders can generate more than six types of silk fibroins, with pyriform silk used to form attachment discs, adhering silk to other surfaces and substances. The unique higher-order structuring of silk fibroins has been cited as the source of their remarkable biomechanical properties. Even so, only one full-length gene sequence of pyriform silk protein 1 (PySp1) from Argiopeargentata has been reported, and studies on the mechanical properties of natural pyriform silk fibers are also lacking. To better understand the PySp1 family of genes, we used long-distance PCR (LD-PCR) to determine the sequence of PySp1 in the Araneusventricosus species. This full-length PySp1 gene is 11,931 bp in length, encoding for 3976 amino acids residues in non-repetitive N- and C-terminal domains with a central largely repetitive region made up of sixteen remarkably homogeneous units. This was similar to the previously reported A. argentata PySp1 sequence, with PySp1 from A. ventricosus also having a long repetitive N-linker that bridges the N-terminal and repetitive regions. Predictions of secondary structure and hydrophobicity of A. ventricosus PySp1 showed the pyriform silk fiber’s functional properties. The amino acid compositions of PySp1 is obviously distinct from other spidroins. Our sequence makes an important contribution to understand pyriform silk protein structure and also provides a new template for recombinant pyriform silk proteins with attractive properties.

First estimation of the scale of canonical 5’ splice site GT>GC mutations generating wild-type transcripts and their medical genetic implications

ABSTRACTIt has long been known that canonical 5’ splice site (5’SS) GT>GC mutations may be compatible with normal splicing. However, to date, the true scale of canonical 5’SS GT>GC mutations generating wild-type transcripts, both in the context of the frequency of such mutations and the level of wild-type transcripts generated from the mutation alleles, remain unknown. Herein, combining data derived from a meta-analysis of 45 informative disease-causing 5’SS GT>GC mutations (from 42 genes) and a cell culture-based full-length gene splicing assay of 103 5’SS GT>GC mutations (from 30 genes), we estimate that ∼15-18% of the canonical GT 5’SSs are capable of generating between 1 and 84% normal transcripts as a consequence of the substitution of GT by GC. We further demonstrate that the canonical 5’SSs whose substitutions of GT by GC generated normal transcripts show stronger complementarity to the 5’ end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild-type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were able to accurately predict the functional impact of 5’SS GT>GC mutations. Our findings imply that 5’SS GT>GC mutations may not invariably cause human disease but should also help to improve our understanding of the evolutionary processes that accompanied GT>GC subtype switching of U2-type introns in mammals.