ResearchPad - dna-construction https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[ArdC, a ssDNA-binding protein with a metalloprotease domain, overpasses the recipient <i>hsdRMS</i> restriction system broadening conjugation host range]]> https://www.researchpad.co/article/elastic_article_7739 Horizontal gene transfer is the main mechanism by which bacteria acquire and disseminate new traits, such as antibiotic resistance genes, that allow adaptation and evolution. Here we identified a gene, ardC, that enables a plasmid to increase its conjugative host range, and thus positively contributes to plasmid fitness. The crystal structure of the antirestriction protein ArdC revealed a fold different from other antirestriction proteins. Our results have wide implications for understanding how a gene enlarges the environments a plasmid can colonize and point to new targets to harness the bacterial DNA uptake control.

]]>
<![CDATA[The mechanism on phosphorylation of Hsp20Ser16 inhibit GA stress and ER stress during OGD/R]]> https://www.researchpad.co/article/5c8acc8cd5eed0c48498f9ce

Recent research has demonstrated that small heat shock protein (sHsp) phosphorylation plays a variety of roles in neural cells. While the phosphorylation of serine 16 (Ser16) is blocked, Hsp20 no longer has neuroprotective effects. To further investigate the mechanism underlying this process, oxygen-glucose deprivation and reperfusion (OGD/R) was used with human SH-SY5Y cells and mouse N2a neuroblastoma cells. When SH-SY5Y and N2a cells were transfected with pEGFP-Hsp20(WT), pEGFP-Hsp20(S16A), and pEGFP-Hsp20(S16D) plasmids, the Golgi apparatus (GA) became more swollen and scattered, and many small fragments formed in the MOCK and S16A groups after OGD/R (P < 0.05). Meanwhile, the endoplasmic reticulum (ER) network was reduced, and the lamellar structure increased. However, these changes were not as obvious in the WT and S16D groups. Additionally, after OGD/R, Golgi Stress related protein contents were increased in the WT and S16D groups compared with the MOCK and S16A groups (P < 0.05). However, ER Stress related protein contents were decreased in the WT and S16D groups compared with the MOCK and S16A groups (P < 0.05). Our study demonstrates that Hsp20 phosphorylation on Ser16 protects against not only OGD/R-induced GA fragmentation in SH-SY5Y cells and N2a cells via Golgi stress but also OGD/R-induced ER structural changes in SH-SY5Y cells via ER stress. These findings suggest that Hsp20 is a potential drug target for ischemia stroke treatment.

]]>
<![CDATA[Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway]]> https://www.researchpad.co/article/5c784fbdd5eed0c484007497

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity.

]]>
<![CDATA[TyrR is involved in the transcriptional regulation of biofilm formation and D-alanine catabolism in Azospirillum brasilense Sp7.]]> https://www.researchpad.co/article/5c6f1503d5eed0c48467ac9f

Azospirillum brasilense is one of the most studied species of diverse agronomic plants worldwide. The benefits conferred to plants inoculated with Azospirillum have been primarily attributed to its capacity to fix atmospheric nitrogen and synthesize phytohormones, especially indole-3-acetic acid (IAA). The principal pathway for IAA synthesis involves the intermediate metabolite indole pyruvic acid. Successful colonization of plants by Azospirillum species is fundamental to the ability of these bacteria to promote the beneficial effects observed in plants. Biofilm formation is an essential step in this process and involves interactions with the host plant. In this study, the tyrR gene was cloned, and the translated product was observed to exhibit homology to TyrR protein, a NtrC/NifA-type activator. Structural studies of TyrR identified three putative domains, including a domain containing binding sites for aromatic amino acids in the N-terminus, a central AAA+ ATPase domain, and a helix-turn-helix DNA binding motif domain in the C-terminus, which binds DNA sequences in promoter-operator regions. In addition, a bioinformatic analysis of promoter sequences in A. brasilense Sp7 genome revealed that putative promoters encompass one to three TyrR boxes in genes predicted to be regulated by TyrR. To gain insight into the phenotypes regulated by TyrR, a tyrR-deficient strain derived from A. brasilense Sp7, named A. brasilense 2116 and a complemented 2116 strain harboring a plasmid carrying the tyrR gene were constructed. The observed phenotypes indicated that the putative transcriptional regulator TyrR is involved in biofilm production and is responsible for regulating the utilization of D-alanine as carbon source. In addition, TyrR was observed to be absolutely required for transcriptional regulation of the gene dadA encoding a D-amino acid dehydrogenase. The data suggested that TyrR may play a major role in the regulation of genes encoding a glucosyl transferase, essential signaling proteins, and amino acids transporters.

]]>
<![CDATA[The genetic intractability of Symbiodinium microadriaticum to standard algal transformation methods]]> https://www.researchpad.co/article/5c75ac68d5eed0c484d08712

Modern transformation and genome editing techniques have shown great success across a broad variety of organisms. However, no study of successfully applied genome editing has been reported in a dinoflagellate despite the first genetic transformation of Symbiodinium being published about 20 years ago. Using an array of different available transformation techniques, we attempted to transform Symbiodinium microadriaticum (CCMP2467), a dinoflagellate symbiont of reef-building corals, with the view to performing subsequent CRISPR-Cas9 mediated genome editing. Plasmid vectors designed for nuclear transformation containing the chloramphenicol resistance gene under the control of the CaMV p35S promoter as well as several putative endogenous promoters were used to test a variety of transformation techniques including biolistics, electroporation and agitation with silicon carbide whiskers. Chloroplast-targeted transformation was attempted using an engineered Symbiodinium chloroplast minicircle encoding a modified PsbA protein expected to confer atrazine resistance. We report that we have been unable to confer chloramphenicol or atrazine resistance on Symbiodinium microadriaticum strain CCMP2467.

]]>
<![CDATA[A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene]]> https://www.researchpad.co/article/5c65dcb0d5eed0c484dec049

Genome-wide association studies have identified more than 200 genetic variants to be associated with an increased risk of developing multiple sclerosis (MS). Still, little is known about the causal molecular mechanisms that underlie the genetic contribution to disease susceptibility. In this study, we investigated the role of the single-nucleotide polymorphism (SNP) rs1414273, which is located within the microRNA-548ac stem-loop sequence in the first intron of the CD58 gene. We conducted an expression quantitative trait locus (eQTL) analysis based on public RNA-sequencing and microarray data of blood-derived cells of more than 1000 subjects. Additionally, CD58 transcripts and mature hsa-miR-548ac molecules were measured using real-time PCR in peripheral blood samples of 32 MS patients. Cell culture experiments were performed to evaluate the efficiency of Drosha-mediated stem-loop processing dependent on genotype and to determine the target genes of this underexplored microRNA. Across different global populations and data sets, carriers of the MS risk allele showed reduced CD58 mRNA levels but increased hsa-miR-548ac levels. We provide evidence that the SNP rs1414273 might alter Drosha cleavage activity, thereby provoking partial uncoupling of CD58 gene expression and microRNA-548ac production from the shared primary transcript in immune cells. Moreover, the microRNA was found to regulate genes, which participate in inflammatory processes and in controlling the balance of protein folding and degradation. We thus uncovered new regulatory implications of the MS-associated haplotype of the CD58 gene locus, and we remind that paradoxical findings can be encountered in the analysis of eQTLs upon data aggregation. Our study illustrates that a better understanding of RNA processing events might help to establish the functional nature of genetic variants, which predispose to inflammatory and neurological diseases.

]]>
<![CDATA[PepN is a non-essential, cell wall-localized protein that contributes to neutrophil elastase-mediated killing of Streptococcus pneumoniae]]> https://www.researchpad.co/article/5c5df336d5eed0c484580f0d

Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause disease in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance of Spn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis, Spn is exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killing Spn, the bacterial proteins that are degraded by NSPs leading to Spn death are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN a CW-localized protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotype in vitro in the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophils ex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing of Spn by NSPs and human neutrophils.

]]>
<![CDATA[Bclaf1 critically regulates the type I interferon response and is degraded by alphaherpesvirus US3]]> https://www.researchpad.co/article/5c57e68bd5eed0c484ef366f

Type I interferon response plays a prominent role against viral infection, which is frequently disrupted by viruses. Here, we report Bcl-2 associated transcription factor 1 (Bclaf1) is degraded during the alphaherpesvirus Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) infections through the viral protein US3. We further reveal that Bclaf1 functions critically in type I interferon signaling. Knockdown or knockout of Bclaf1 in cells significantly impairs interferon-α (IFNα) -mediated gene transcription and viral inhibition against US3 deficient PRV and HSV-1. Mechanistically, Bclaf1 maintains a mechanism allowing STAT1 and STAT2 to be efficiently phosphorylated in response to IFNα, and more importantly, facilitates IFN-stimulated gene factor 3 (ISGF3) binding with IFN-stimulated response elements (ISRE) for efficient gene transcription by directly interacting with ISRE and STAT2. Our studies establish the importance of Bclaf1 in IFNα-induced antiviral immunity and in the control of viral infections.

]]>
<![CDATA[CDI/CDS system-encoding genes of Burkholderia thailandensis are located in a mobile genetic element that defines a new class of transposon]]> https://www.researchpad.co/article/5c3d00f6d5eed0c484037189

Intercellular communication and self-recognition are critical for coordinating cooperative and competitive behaviors during sociomicrobiological community development. Contact-dependent growth inhibition (CDI) proteins are polymorphic toxin delivery systems that inhibit the growth of non-self neighboring bacteria that lack the appropriate immunity protein. In Burkholderia thailandensis, CDI system proteins (encoded by bcpAIOB genes) also induce cooperative behaviors among sibling (self) cells, a phenomenon called contact-dependent signaling (CDS). Here we describe a mobile genetic element (MGE) that carries the bcpAIOB genes in B. thailandensis E264. It is a ~210 kb composite transposon with insertion sequence (IS) elements at each end. Although the ISs are most similar to IS2 of Escherichia coli, the transposase-dependent intermediate molecule displays characteristics more similar to those of the IS26 translocatable unit (TU). A reaction requiring only the “left” IS-encoded transposase results in formation of an extrachromosomal circular dsDNA intermediate (“the megacircle”) composed of the left IS and the sequences intervening between the ISs. Insertion of the megacircle into the chromosome occurs next to a pre-existing copy of an IS2-like element, recreating a functional composite transposon. We found that BcpA activity is required for megacircle formation, and in turn, megacircle formation is required for CDS phenotypes. Our data support a model in which the bcpAIOB genes function as both helping and harming greenbeard genes, simultaneously enhancing the fitness of self bacteria that possess the same allele plus tightly linked genes that mediate cooperative behaviors, and killing non-self bacteria that do not possess the same bcpAIOB allele. Mobility of the megacircle between cells could allow bacteria invading a community to be converted to self, and would facilitate propagation of the bcpAIOB genes in the event that the invading strain is capable of overtaking the resident community.

]]>
<![CDATA[Discovery of Leptospira spp. seroreactive peptides using ORFeome phage display]]> https://www.researchpad.co/article/5c536b32d5eed0c484a482f9

Background

Leptospirosis is the most common zoonotic disease worldwide. The diagnostic performance of a serological test for human leptospirosis is mainly influenced by the antigen used in the test assay. An ideal serological test should cover all serovars of pathogenic leptospires with high sensitivity and specificity and use reagents that are relatively inexpensive to produce and can be used in tropical climates. Peptide-based tests fulfil at least the latter two requirements, and ORFeome phage display has been successfully used to identify immunogenic peptides from other pathogens.

Methodology/Principal findings

Two ORFeome phage display libraries of the entire Leptospira spp. genomes from five local strains isolated in Malaysia and seven WHO reference strains were constructed. Subsequently, 18 unique Leptospira peptides were identified in a screen using a pool of sera from patients with acute leptospirosis. Five of these were validated by titration ELISA using different pools of patient or control sera. The diagnostic performance of these five peptides was then assessed against 16 individual sera from patients with acute leptospirosis and 16 healthy donors and was compared to that of two recombinant reference proteins from L. interrogans. This analysis revealed two peptides (SIR16-D1 and SIR16-H1) from the local isolates with good accuracy for the detection of acute leptospirosis (area under the ROC curve: 0.86 and 0.78, respectively; sensitivity: 0.88 and 0.94; specificity: 0.81 and 0.69), which was close to that of the reference proteins LipL32 and Loa22 (area under the ROC curve: 0.91 and 0.80; sensitivity: 0.94 and 0.81; specificity: 0.75 and 0.75).

Conclusions/Significance

This analysis lends further support for using ORFeome phage display to identify pathogen-associated immunogenic peptides, and it suggests that this technique holds promise for the development of peptide-based diagnostics for leptospirosis and, possibly, of vaccines against this pathogen.

]]>
<![CDATA[Interaction between the transmembrane domains of Sho1 and Opy2 enhances the signaling efficiency of the Hog1 MAP kinase cascade in Saccharomyces cerevisiae]]> https://www.researchpad.co/article/5c57e68cd5eed0c484ef36b5

To cope with increased extracellular osmolarity, the budding yeast Saccharomyces cerevisiae activates the Hog1 mitogen-activated protein kinase (MAPK), which controls a variety of adaptive responses. Hog1 is activated through the high-osmolarity glycerol (HOG) pathway, which consists of a core MAPK cascade and two independent upstream branches (SHO1 and SLN1 branches) containing distinct osmosensing machineries. In the SHO1 branch, a homo-oligomer of Sho1, the four-transmembrane (TM) osmosensor, interacts with the transmembrane co-osmosensors, Hkr1 and Msb2, and the membrane anchor protein Opy2, through their TM domains, and activates the Ste20-Ste11-Pbs2-Hog1 kinase cascade. In this study, we isolated and analyzed hyperactive mutants of Sho1 and Opy2 that harbor mutations within their TM domains. Several hyperactive mutations enhanced the interaction between Sho1 and Opy2, indicating the importance of the TM-mediated interaction between Sho1 and Opy2 for facilitating effective signaling. The interaction between the TM domains of Sho1 and Opy2 will place their respective cytoplasmic binding partners Pbs2 and Ste11 in close proximity. Indeed, genetic analyses of the mutants showed that the Sho1-Opy2 interaction enhances the activation of Pbs2 by Ste11, but not Hog1 by Pbs2. Some of the hyperactive mutants had mutations at the extracellular ends of either Sho1 TM4 or Opy2 TM, and defined the Sho1-Opy2 binding site 1 (BS1). Chemical crosslinking and mutational analyses revealed that the cytoplasmic ends of Sho1 TM1 and Opy2 TM also interact with each other, defining the Sho1-Opy2 binding site 2 (BS2). A geometric consideration constrains that one Opy2 molecule must interact with two adjacent Sho1 molecules in Sho1 oligomer. These results raise a possibility that an alteration of the conformation of the Sho1-Opy2 complex might contributes to the osmotic activation of the Hog1 MAPK cascade.

]]>
<![CDATA[Asymmetric diversification of mating pheromones in fission yeast]]> https://www.researchpad.co/article/5c50c46fd5eed0c4845e875d

In fungi, mating between partners depends on the molecular recognition of two peptidyl mating pheromones by their respective receptors. The fission yeast Schizosaccharomyces pombe (Sp) has two mating types, Plus (P) and Minus (M). The mating pheromones P-factor and M-factor, secreted by P and M cells, are recognized by the receptors mating type auxiliary minus 2 (Mam2) and mating type auxiliary plus 3 (Map3), respectively. Our recent study demonstrated that a few mutations in both M-factor and Map3 can trigger reproductive isolation in S. pombe. Here, we explored the mechanism underlying reproductive isolation through genetic changes of pheromones/receptors in nature. We investigated the diversity of genes encoding the pheromones and their receptor in 150 wild S. pombe strains. Whereas the amino acid sequences of M-factor and Map3 were completely conserved, those of P-factor and Mam2 were very diverse. In addition, the P-factor gene contained varying numbers of tandem repeats of P-factor (4–8 repeats). By exploring the recognition specificity of pheromones between S. pombe and its close relative Schizosaccharomyces octosporus (So), we found that So-M-factor did not have an effect on S. pombe P cells, but So-P-factor had a partial effect on S. pombe M cells. Thus, recognition of M-factor seems to be stringent, whereas that of P-factor is relatively relaxed. We speculate that asymmetric diversification of the two pheromones might be facilitated by the distinctly different specificities of the two receptors. Our findings suggest that M-factor communication plays an important role in defining the species, whereas P-factor communication is able to undergo a certain degree of flexible adaptation–perhaps as a first step toward prezygotic isolation in S. pombe.

]]>
<![CDATA[Mutant T4 DNA polymerase for easy cloning and mutagenesis]]> https://www.researchpad.co/article/5c5217bad5eed0c484794479

The advent of high-fidelity DNA polymerases that can be used to linearize and amplify whole plasmids by PCR opened the door to greatly simplified cloning and mutagenesis protocols. Commercially available kits work well, but often have been optimized using undisclosed or proprietory components. Here we show that a mutant T4 DNA polymerase (Y320A) with attenuated 3’-exonuclease activity is uniquely suited to generate single-stranded DNA overhangs of uniform length in a more easily controllable manner than the wild-type enzyme, and this can be used to increase the yields of colonies containing correctly modified plasmids in cloning and mutagenesis experiments, which is particularly useful when E. coli cells are of relatively low competency. Standard protocols using the mutant T4 DNA polymerase are provided for the sequence and ligation independent cloning (SLIC) method and a modified QuikChange method, where the mutant enzyme enhances the yield of correctly mutated plasmid and further suppresses parental plasmid during digestion with DpnI. Single-stranded DNA overhangs generated by the mutant T4 DNA polymerase facilitate subsequent plasmid circularization, annealing and ligation in E. coli.

]]>
<![CDATA[Functional screening for triclosan resistance in a wastewater metagenome and isolates of Escherichia coli and Enterococcus spp. from a large Canadian healthcare region]]> https://www.researchpad.co/article/5c536bccd5eed0c484a49249

The biocide triclosan is in many consumer products and is a frequent contaminant of wastewater (WW) such that there is concern that triclosan promotes resistance to important antibiotics. This study identified functional mechanisms of triclosan resistance (TCSR) in WW metagenomes, and assessed the frequency of TCSR in WW-derived and clinical isolates of Escherichia coli and Enterococcus spp. Metagenomic DNA extracted from WW was used to profile the microbiome and construct large-insert cosmid libraries, which were screened for TCSR. Resistant cosmids were sequenced and the TCSR determinant identified by transposon mutagenesis. Wastewater Enterococcus spp. (N = 94) and E. coli (N = 99) and clinical Enterococcus spp. (N = 146) and vancomycin-resistant E. faecium (VRE; N = 149) were collected and tested for resistance to triclosan and a comprehensive drug panel. Functional metagenomic screening revealed diverse FabV homologs as major WW TCSR determinants. Resistant clones harboured sequences likely originating from Aeromonas spp., a common WW microbe. The triclosan MIC90s for E. coli, E. faecalis, and E. faecium isolates were 0.125, 32, and 32 mg/L, respectively. For E. coli, there was no correlation between the triclosan MIC and any drug tested. Negative correlations were detected between the triclosan MIC and levofloxacin resistance for E. faecalis, and between triclosan and vancomycin, teicoplanin, and ampicillin resistance for E. faecium. Thus, FabV homologs were the major contributor to the WW triclosan resistome and high-level TCSR was not observed in WW or clinical isolates. Elevated triclosan MICs were not positively correlated with antimicrobial resistance to any drug tested.

]]>
<![CDATA[Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580]]> https://www.researchpad.co/article/5c478c3bd5eed0c484bd0f6c

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

]]>
<![CDATA[CRISPR-Cas9 In Situ engineering of subtilisin E in Bacillus subtilis]]> https://www.researchpad.co/article/5c3d0133d5eed0c48403922d

CRISPR-Cas systems have become widely used across all fields of biology as a genome engineering tool. With its recent demonstration in the Gram positive industrial workhorse Bacillus subtilis, this tool has become an attractive option for rapid, markerless strain engineering of industrial production hosts. Previously described strategies for CRISPR-Cas9 genome editing in B. subtilis have involved chromosomal integrations of Cas9 and single guide RNA expression cassettes, or construction of large plasmids for simultaneous transformation of both single guide RNA and donor DNA. Here we use a flexible, co-transformation approach where the single guide RNA is inserted in a plasmid for Cas9 co-expression, and the donor DNA is supplied as a linear PCR product observing an editing efficiency of 76%. This allowed multiple, rapid rounds of in situ editing of the subtilisin E gene to incorporate a salt bridge triad present in the Bacillus clausii thermotolerant homolog, M-protease. A novel subtilisin E variant was obtained with increased thermotolerance and activity.

]]>
<![CDATA[Imputation accuracy of wheat genotyping-by-sequencing (GBS) data using barley and wheat genome references]]> https://www.researchpad.co/article/5c3d0126d5eed0c484038b91

Genotyping-by-sequencing (GBS) provides high SNP coverage and has recently emerged as a popular technology for genetic and breeding applications in bread wheat (Triticum aestivum L.) and many other plant species. Although GBS can discover millions of SNPs, a high rate of missing data is a major concern for many applications. Accurate imputation of those missing data can significantly improve the utility of GBS data. This study compared imputation accuracies among four genome references including three wheat references (Chinese Spring survey sequence, W7984, and IWGSC RefSeq v1.0) and one barley reference genome by comparing imputed data derived from low-depth sequencing to actual data from high-depth sequencing. After imputation, the average number of imputed data points was the highest in the B genome (~48.99%). The D genome had the lowest imputed data points (~15.02%) but the highest imputation accuracy. Among the four reference genomes, IWGSC RefSeq v1.0 reference provided the most imputed data points, but the lowest imputation accuracy for the SNPs with < 10% minor allele frequency (MAF). The W7984 reference, however, provided the highest imputation accuracy for the SNPs with < 10% MAF.

]]>
<![CDATA[Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo]]> https://www.researchpad.co/article/5c141f0dd5eed0c484d296ca

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.

]]>
<![CDATA[CDHR3 extracellular domains EC1-3 mediate rhinovirus C interaction with cells and as recombinant derivatives, are inhibitory to virus infection]]> https://www.researchpad.co/article/5c1813ced5eed0c484775dec

Viruses in the rhinovirus C species (RV-C) are more likely to cause severe wheezing illnesses and asthma exacerbations in children than related isolates of the RV-A or RV-B. The RV-C capsid is structurally distinct from other rhinoviruses and does not bind ICAM-1 or LDL receptors. The RV-C receptor is instead, human cadherin-related family member 3 (CDHR3), a protein unique to the airway epithelium. A single nucleotide polymorphism (rs6967330, encoding C529Y) in CDHR3 regulates the display density of CDHR3 on cell surfaces and is among the strongest known genetic correlates for childhood virus-induced asthma susceptibility. CDHR3 immunoprecipitations from transfected or transduced cell lysates were used to characterize the RV-C interaction requirements. The C529 and Y529 variations in extracellular repeat domain 5 (EC5), bound equivalently to virus. Glycosylase treatment followed by mass spectrometry mapped 3 extracellular N-linked modification sites, and further detected surface-dependent, α2–6 sialyation unique to the Y529 format. None of these modifications were required for RV-C recognition, but removal or even dilution of structurally stabilizing calcium ions from the EC junctions irreversibly abrogated virus binding. CDHR3 deletions expressed in HeLa cells or as bacterial recombinant proteins, mapped the amino-terminal EC1 unit as the required virus contact. Derivatives containing the EC1 domain, could not only recapitulate virus:receptor interactions in vitro, but also directly inhibit RV-C infection of susceptible cells for several virus genotypes (C02, C15, C41, and C45). We propose that all RV-C use the same EC1 landing pad, interacting with putative EC3-mediated multimerization formats of CDHR3.

]]>
<![CDATA[BLM prevents instability of structure-forming DNA sequences at common fragile sites]]> https://www.researchpad.co/article/5c099406d5eed0c4842ae0ea

Genome instability often arises at common fragile sites (CFSs) leading to cancer-associated chromosomal rearrangements. However, the underlying mechanisms of how CFS protection is achieved is not well understood. We demonstrate that BLM plays an important role in the maintenance of genome stability of structure-forming AT-rich sequences derived from CFSs (CFS-AT). BLM deficiency leads to increased DSB formation and hyper mitotic recombination at CFS-AT and induces instability of the plasmids containing CFS-AT. We further showed that BLM is required for suppression of CFS breakage upon oncogene expression. Both helicase activity and ATR-mediated phosphorylation of BLM are important for preventing genetic instability at CFS-AT sequences. Furthermore, the role of BLM in protecting CFS-AT is not epistatic to that of FANCM, a translocase that is involved in preserving CFS stability. Loss of BLM helicase activity leads to drastic decrease of cell viability in FANCM deficient cells. We propose that BLM and FANCM utilize different mechanisms to remove DNA secondary structures forming at CFS-AT on replication forks, thereby preventing DSB formation and maintaining CFS stability.

]]>