Rapid Amplification Of cDNA Ends Research Articles

Local aromatase excess with recruitment of unusual promoters of CYP19A1 gene in prepubertal patients with gynecomastia.

Gynecomastia may be due to aromatase excess in several diseases such as obesity and cancer. Aromatase excess syndrome (AEXS) is an autosomal dominant disorder caused by overexpression of CYP19A1. Germinal mutations occurring in AEXS include various genomic rearrangements including duplication, deletion, and inversion identified in the upstream region of CYP19A1. Aromatase overexpression caused by a CYP19A1 somatic mutation has been rarely described. Breast adipose tissue biopsies or surgical specimens were obtained from 19 subjects with gynecomastia. Aromatase quantification was performed by digital PCR and CYP19A1 sequencing by RACE PCR products. We observed localized aromatase overexpression (>10 fold greater than normal) in breast adipose tissue from three prepubertal males with gynecomastia out of the 19 cases. One carried a chromosomal rearrangement between CYP19A1 and DMXL2, consistent with AEXS. In the 2 others, the first exon of CYP19A1 contained 11 different tissue-specific promoter subtypes, specifically I.4 or I.3 normally expressed by adipose tissue, but also the placental I.2 promoter and the more ubiquitous I.7 which is usually expressed in breast cancer, uterine, and endothelial tissues. No differences in clinical or biochemical characteristics were observed between these 3 subjects and 16 others without aromatase overexpression. We describe two cases of aromatase overexpression in breast adipose tissue associated with nonspecific promoter recruitment. Further investigations are necessary to understand the mechanisms involved in aberrant promoter selection.

Identification and Functional Analysis of the G1 Phase Cyclin Dependent Kinase Gene Hc-CDK6 in Pearl Mussels (Hyriopsis cumingii)

Cyclin dependent kinase 6 (CDK6) is a serine/threonine kinase that plays important roles in cell cycle progression and differentiation. In this study, full-length cDNA of Hc-CDK6 was obtained from freshwater pearl mussels (Hyriopsis cumingii, Hc) with 3´,5´ rapid-amplification of cDNA ends (RACE). The Hc-CDK6 expression profiles were analyzed with quantitative real-time PCR and in situ hybridization. The function of the Hc-CDK6 gene was studied with both RNA interference (RNAi) and overexpression in H. cumingii. Hc-CDK6 was found to encode 331 amino acids and to have a CDK4/6-like serine/threonine kinase catalytic structural domain. In terms of the amino acid sequence, the protein Hc-CDK6 was most closely related to its homolog in Crassostrea gigas, with a similarity of 75.23%. Hc-CDK6 was expressed in all examined tissues (adductor, foot, visceral mass, gill, outer mantle, inner mantle and gonads), and the highest expression was observed in the gonads (P<0.05). The relative expression of Hc-CDK6 increased during embryonic development, and was higher in the blastocyst and gastrulation stages, which were characterized by rapid division and differentiation. Hc-CDK6 showed hybridization signals in all parts of the mantle. After knockdown of Hc-CDK6 through RNAi, a significant decrease in CDK6 expression was found, and the percentage of cells in G0/G1 significantly increased. Overexpression of Hc-CDK6 in mantle cells increased the proliferation of cultured cells (P<0.05). Hc-CDK6 appeared to promote the cell cycle in H. cumingii, and overexpression of Hc-CDK6 promoted mantle cell proliferation. The functional study of this gene may provide new ideas for solving the problem of slow proliferation of shellfish cells in in vitro culture.

Full genome sequence of a novel iflavirus from the leafhopper Recilia dorsalis.

The leafhopper Recilia dorsalis (family Cicadellidae, tribe Deltocephalini) is a common pest of rice and a transmitter of various viruses. Here, we discovered a novel virus in an R. dorsalis sample and determined its complete genome sequence by metagenomic sequencing and rapid amplification of cDNA ends. Based on a homology search and phylogenetic analysis, we show that the new virus belongs to the genus Iflavirus, family Iflaviridae, and we have tentatively named it "Recilia dorsalis iflavirus 1" (RdIV1). Excluding the polyA tail, the RdIV1 genome is 10,986 nucleotides in length and is predicted to encode a 3,195-amino-acid-long polyprotein that possesses the typical domains of iflaviruses: two rhinovirus-like (rhv-like) capsid domains, a cricket paralysis virus-like (CRPV-like) capsid domain, a helicase domain, a protease domain, and an RNA-dependent RNA polymerase (RdRp) domain. BLAST searches showed that the RdIV1 genome has the highest amino sequence identity (73.8%) in the coat protein region to Euscelidiusvariegatusvirus1 (EVV-1), a member of to the genus Iflavirus, indicating that RdIV1 can be classified as a new iflavirus.

Two Novel Iflaviruses Discovered in Bat Samples in Washington State.

Arthropods are integral to ecosystem equilibrium, serving as both a food source for insectivores and supporting plant reproduction. Members of the Iflaviridae family in the order Picornavirales are frequently found in RNA sequenced from arthropods, who serve as their hosts. Here we implement a metagenomic deep sequencing approach followed by rapid amplification of cDNA ends (RACE) on viral RNA isolated from wild and captured bat guano in Washington State at two separate time points. From these samples we report the complete genomes of two novel viruses in the family Iflaviridae. The first virus, which we call King virus, is 46% identical by nucleotide to the lethal honeybee virus, deformed wing virus, while the second virus which we call Rolda virus, shares 39% nucleotide identity to deformed wing virus. King and Rolda virus genomes are 10,183 and 8934 nucleotides in length, respectively. Given these iflaviruses were detected in guano from captive bats whose sole food source was the Tenebrio spp. mealworm, we anticipate this invertebrate may be a likely host. Using the NCBI Sequence Read Archive, we found that these two viruses are located in six continents and have been isolated from a variety of arthropod and mammalian specimens.

Construction and Characterization of a Botrytis Virus F Infectious Clone.

Botrytis virus F (BVF) is a positive-sense, single-stranded RNA (+ssRNA) virus within the Gammaflexiviridae family of the plant-pathogenic fungus Botrytis cinerea. In this study, the complete sequence of a BVF strain isolated from B. cinerea collected from grapevine fields in Spain was analyzed. This virus, in this work BVF-V448, has a genome of 6827 nt in length, excluding the poly(A) tail, with two open reading frames encoding an RNA dependent RNA polymerase (RdRP) and a coat protein (CP). The 5′- and 3′-terminal regions of the genome were determined by rapid amplification of cDNA ends (RACE). Furthermore, a yet undetected subgenomic RNA species in BVF-V448 was identified, indicating that the CP is expressed via 3′ coterminal subgenomic RNAs (sgRNAs). We also report the successful construction of the first BVF full-length cDNA clone and synthesized in vitro RNA transcripts using the T7 polymerase, which could efficiently transfect two different strains of B. cinerea, B05.10 and Pi258.9. The levels of growth in culture and virulence on plants of BVF-V448 transfected strains were comparable to BVF-free strains. The infectious clones generated in this work provide a useful tool for the future development of an efficient BVF foreign gene expression vector and a virus-induced gene silencing (VIGS) vector as a biological agent for the control of B. cinerea.

Complete genome sequence of Amazon lily mosaic virus isolated from amaryllis (Hippeastrum hybridum Hort.).

In April 2011, a virus was isolated by single-lesion isolation on Chenopodium quinoa leaves from an amaryllis plant with chlorotic ringspots in a private garden in Changhua County, Taiwan. An Illumina MiSeq sequencing system was used to determine the genomic nucleotide (nt) sequence of the virus. A de novo-assembled contig with 9377 nt, containing an open reading frame encoding a putative potyviral polyprotein, was annotated as the potyvirus Amazon lily mosaic virus (ALiMV), sharing 95.5% nt sequence identity with a partial genomic sequence of ALiMV available in the GenBank database. Therefore, the amaryllis virus was designated as ALiMV-TW. Through 5´ and 3´ rapid amplification of cDNA ends (RACE), the complete 9618-nt genome sequence of ALiMV-TW was determined. Sequence comparisons indicated that the genome and polyprotein of ALiMV-TW share 52.3-65.1% nt and 30.1-64.2% aa sequence identity, respectively, with those of other potyviruses. This is the first report of a complete genome sequence of ALiMV.

CsbZIP2-miR9748-CsNPF4.4 Module Mediates High Temperature Tolerance of Cucumber Through Jasmonic Acid Pathway.

High temperature stress seriously affects the growth of cucumber seedlings, and even leads to a decline in yield and quality. miRNAs have been shown to be involved in regulating the response to stress in plants, but little is known about its effects on cucumber high temperature stress tolerance. Here, we found that high temperature stress induced the expression of miR9748 in cucumber. Overexpression of cucumber miR9748 in Arabidopsis improved high temperature tolerance. Transcriptome analysis revealed that miR9748 might mediate high temperature tolerance through plant hormone signal pathway. 5′ RNA ligase-mediated rapid amplification of cDNA ends (5′ RLM-RACE) and transient transformation technology demonstrated that CsNPF4.4 was the target gene of miR9748. CsNPF4.4 overexpression plants decreased high temperature tolerance accompanied by reducing the content of jasmonic acid (JA), but alleviated by foliar application of methyl jasmonate, indicating that CsNPF4.4 negatively regulated high temperature stress tolerance through inhibition JA signal pathway. Furthermore, high temperature stress also increased the expression level of CsbZIP2. Yeast one-hybrid and dual-luciferase assays showed that CsbZIP2 directly bound to the promoter of MIR9748 to induce its expression. Taken together, our results indicated that CsbZIP2 directly regulated miR9748 expression to cleave CsNPF4.4 to mediate high temperature tolerance through JA pathway.

Draft Genome Sequence of a Novel Iflavirus from Leafhoppers (Exitianus capicola) in Iraq.

ABSTRACTThe draft genome sequence of a novel virus was determined from a leafhopper insect (Exitianus capicola) via transcriptomic sequencing and rapid amplification of cDNA ends. Results of the sequence alignment suggested that the new virus is a member of the genus Iflavirus, family Iflaviridae. Thus, it was proposed to be named Exitianus capicola iflavirus 2 (ECIV2). ECIV2 has a single positive-strand RNA genome of 7,821 nucleotides [excluding the poly(A) tail] containing a single open reading frame (ORF) with typical iflavirus conserved domains, including a picornavirus capsid protein-like domain, a cricket paralysis virus capsid-like domain, a helicase, a peptidase, and an RNA-dependent RNA polymerase domain.

Genome-Wide Investigation of the MiR166 Family Provides New Insights into Its Involvement in the Drought Stress Responses of Tea Plants (Camellia sinensis (L.) O. Kuntze)

MicroRNA166 (miR166) is a highly conserved plant miRNA that plays a crucial role in plant growth and the resistance to various abiotic stresses. However, the miR166s in tea (Camellia sinensis (L.) O. Kuntze) have not been comprehensively identified and analyzed. This study identified 30 mature miR166s and twelve pre-miR166s in tea plants. An evolutionary analysis revealed that csn-miR166s originating from the 3′ arm of their precursors were more conserved than the csn-miR166s derived from the 5′ arm of their precursors. The twelve pre-miR166s in tea were divided into two groups, with csn-MIR166 Scaffold364-2 separated from the other precursors. The Mfold-based predictions indicated that the twelve csn-MIR166s formed typical and stable structures comprising a stem-loop hairpin, with minimum free energy ranging from −110.90 to −71.80 kcal/mol. An analysis of the CsMIR166 promoters detected diverse cis-acting elements, including those related to light responses, biosynthesis and metabolism, abiotic stress defenses, and hormone responses. There was no one-to-one relationship between the csn-miR166s and their targets, but most csn-miR166s targeted HD-Zip III genes. Physiological characterization of tea plants under drought stress showed that leaf water content proportionally decreased with the aggravation of drought stress. In contrast, tea leaves’ malondialdehyde (MDA) content proportionally increased. Moreover, the cleavage site of the ATHB-15-like transcript was identified according to a modified 5′ RNA ligase-mediated rapid amplification of cDNA ends. The RT-qPCR data indicated that the transcription of nine csn-miR166s was negatively correlated with their target gene.

Molecular cloning, characterization and expression analysis of P53 from high latitude fish Phoxinus lagowskii and its response to hypoxia.

As an intermediate link between multiple cellular stresses and cellular responses, p53, together with its upstream and downstream regulators and related genes, constitutes a complex network that regulates cellular stresses and cellular responses. However, no studies have investigated p53 in Phoxinus lagowskii, particularly the expression of p53 under different hypoxic conditions. In the present study, the cDNA of p53 from the Phoxinus lagowskii was cloned by the combination of homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of Pl-p53 was 1878bp, including an open reading frame (ORF) of 1116bp encoding a polypeptide of 371 amino acids with a predicted molecular weight of 41.22kDa and a theoretical isoelectric point of 7.38. Quantitative real-time (qRT) PCR assays revealed that Pl-p53 was commonly expressed in all tissues examined, with highest expression in the heart. In addition, we investigated the expression of Pl-p53 in different tissues under different hypoxic conditions. In the short-term hypoxia group, Pl-p53 expression was down-regulated in both the brain and heart. The Pl-p53 expression was significantly elevated at 6h in the muscle and liver, and was significantly up-regulated at 24h in spleen. These results suggest that Pl-p53 plays different regulatory roles and provide a theoretical basis for the changes of p53 in fish facing hypoxic environments.

Chicken CDS2 isoforms presented distinct spatio-temporal expression pattern and regulated by insulin in a breed-specific manner

The cytidine diphosphate diacylglycerol synthases (CDSs) gene encodes the cytidine diphosphate-diacylglycerol (CDP-DAG) synthase enzyme that catalyzes the formation of CDP-diacylglycerol from phosphatidic acid. At present, there are no reports of CDS2 in birds. Here, we identified chicken CDS2 transcripts by combining conventional RT-PCR amplification, 5′ rapid amplification of cDNA ends (RACE), and 3′ RACE, explored the spatio-temporal expression profiles of total CDS2 and the longest transcript variant CDS2-4, and investigated the effect of exogenous insulin on the mRNA level of total CDS2 via quantitative RT-PCR. Four transcripts of chicken CDS2 (CDS2-1, -2, -3, and -4) were identified, which were alternatively spliced at the 3′-untranslated region (UTR). Both total CDS2 and CDS2-4 were prominently expressed in adipose tissue, and exhibited low expression in liver and pectoralis of 49-day-old chickens. Regarding the spatio-temporal expression patterns of CDS2 in chicken, total CDS2 exhibited a similar temporal expression tendency with a high level in the later period of incubation (embryonic day 19 [E19] or 1-day-old) in the brain, liver, and pectoralis. While CDS2-4 presented a distinct temporal expression pattern in these tissues, CDS2-4 levels peaked at 21 d in the brain and pectoralis, while liver CDS2-4 mRNA levels were highest at the early stage of hatching (E10). Total CDS2 (P < 0.001) and CDS2-4 (P = 0.0090) mRNA levels in the liver were differentially regulated throughout the development of the chicken. Total CDS2 levels in the liver of Silky chickens were higher than that of the broiler in the basal state and after insulin stimulation. Exogenous insulin significantly down-regulated the level of total CDS2 at 240 min in the pectoralis of Silky chickens (P < 0.01). In conclusion, chicken CDS2 isoforms with variation at the 3′-UTR were identified, which was prominently expressed in adipose tissue. Total CDS2 and CDS2-4 presented distinct spatio-temporal expression patterns, that is they were differentially regulated with age in brain, liver, and pectoralis. Insulin could regulate chicken CDS2 levels in a breed- and tissue-specific manner.

Onchidium reevesii may be able to distinguish low-frequency sound to discriminate the state of tides

ABSTRACT In order to study how Onchidium reevesii predict tides, the cDNA encoding the precursor of the pedal peptide (Pep), a neuropeptide that plays an important role in neuromotor regulation, was cloned by RACE (Rapid Amplification of cDNA Ends) technology. Bioinformatics analysis was carried out on the sequence of the cloned cDNA. The frequency spectra of the intertidal zone were obtained and Quantitative Real-time PCR (qRT-PCR) experiments were carried out to study the connection between low-frequency sound and tidal perception. The full length of the cDNA was 1627 bp and it encodes 15 copies of Pep; The frequency spectra and qRT-PCR results suggest that O. reevesii can distinguish low-frequency sound waves (100–130 Hz) produced by tides, enabling them to avoid tidal immersion or to come out to feed after the tides falls. In this species, Pep may act as the neurotransmitter for tidal perception based on low-frequency sound signals. These experiments will provide a reference for the further study of the mechanism by which O. reevesii senses tides.

PcVDAC promotes WSSV infection by regulating the apoptotic activity of haemocytes in Procambarus clarkii.

Apoptosis is programmed cell death that is strictly regulated by a series of related genes and is of great importance in resisting pathogen invasion and maintaining cell environment homeostasis. Among apoptotic proteins, the voltage-dependent anion channel protein (VDAC) plays a key role in the mitochondrial apoptosis pathway because of its close connection with changes in mitochondrial membrane potential. However, the role of VDAC in apoptosis and immune regulation in Procambarus clarkii is poorly understood. In this study, the VDAC gene in P. clarkii (PcVDAC) was cloned by rapid amplification of cDNA ends (RACE) technology. The gene was found to have a total length of 2277bp, including a 194-bp 5'-UTR, 1234-bp 3'-UTR and 849-bp open reading frame (ORF), and to encode 282 amino acids. PcVDAC was expressed in all tissues tested, and its expression was upregulated after white spot syndrome virus (WSSV) infection (P<0.05). The RNA interference (RNAi) method was used to explore the role of PcVDAC in WSSV infection. The results showed that the number of WSSV copies in haemocytes was significantly reduced after RNAi (P<0.05), and the survival rate was significantly increased. In addition, after RNAi, the apoptosis rate was significantly increased (P<0.05), the mitochondrial membrane potential was reduced (P<0.01), and the expression of caspase-3 and other genes was upregulated (P<0.05). These results indicate that PcVDAC promotes the replication of WSSV in P. clarkii by inhibiting haemocytes apoptosis. Therefore, the results presented in this paper provide new insights into the immune response of P. clarkii infected with WSSV.

Some novel features of strong promoters discovered in Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an important Gram-negative bacterium belonging to the Bacteroides phylum that can efficiently degrade cellulose. But the promoter that mediates the initiation of gene transcription has been unknown for a long time. In this study, we determined the transcription start site (TSS) of C. hutchinsonii by 5' rapid amplification of cDNA ends (5'RACE). The promoter structure was first identified as TAAT and TATTG which are located -5 and -31bp upstream of TSS, respectively. The function of -5 and -31 regions and the spacer length of the promoter Pchu_1284 were explored by site directed ligase-independent mutagenesis (SLIM). The results showed that the promoter activities were sharply decreased when the TTG motif was mutated into guanine (G) or cytosine (C). Interestingly, we found that the strong promoter was accompanied with many TTTG motifs which could enhance the promoter activities within certain copies. These characteristics were different from other promoters of Bacteriodes species. Furthermore, we carried out genome scanning analysis for C. hutchinsonii and another Bacteroides species by Perl6.0. The results indicated that the promoter structure of C. hutchinsonii possessed more unique features than other species. Also, the screened inducible promoter Pchu_2268 was used to overexpress protein CHU_2196 with a molecular weight of 120kDa in C. hutchinsonii. The present study enriched the promoter structure of Bacteroidetes species and also provided a novel method for the highly expressed large protein (cellulase) in vivo, which was helpful to elucidate the unique cellulose degradation mechanism of C. hutchinsonii.Key points• The conserved structure of strong promoter of C. hutchinsonii was elucidated.• Two novel regulation motifs of TTTG and AATTATG in the promoter were discovered.• A new method for induced expression of cellulase in vivo was established.• Helpful for explained the unique cellulose degradation mechanism of C. hutchinsonii.

First Report of a Secovirus Associated with Mountain Celery Chlorotic Spot Disease in Heilongjiang, China.

Mountain celery (Heracleum moellendorffii Hance), an edible perennial herb of Northeast Asia, is sporadically cultivated as a vegetable crop or for medicinal purposes in Northeast China and Korea [1]. In July 2019, a small field of mountain celeries showing chlorotic spots was found in Wangkui, Heilongjiang, China. A small-RNA (sRNA) library was constructed with equal amounts of leaf tissues of a diseased mountain celery and a tomato sample showing mottling symptom from a nearby field using the TruSeq small RNA library preparation kit (Illumina). The library was sequenced by the HiSeq 4000 sequencer at Lianchuan Biotechnology Co., Ltd (Hangzhou, China). After trimming adaptor sequences and discarding low quality reads by Cutadapt [2], the remaining 6,949,946 reads of 17 to 27 nucleotides (nt) were de novo assembled as described [3]. The resulting 395 contigs were searched against the GenBank viral sequence database using the BLASTn and BLASTx algorithms. Twenty-three contigs showed high nt sequence similarities (89-100%) to the genomic sequence of tomato mosaic virus (ToMV). The deduced amino acid (aa) sequences of thirty contigs had 22-96% aa sequence identities to viruses in the family Secoviridae, e.g., surrounding non-legume associated secovirus (snLaSV) and lettuce secovirus 1 (LSV-1). No contig homologous to the genomic sequences of other plant viruses was identified. Total RNAs were extracted from the mountain celery and tomato separately and reverse transcribed into cDNAs by random hexamer plus Oligo-dT(18) using the Super® IV Reverse Transcriptase (Invitrogen, Shanghai, China). Polymerase chain reactions (PCR) showed that the secovirus was derived from the mountain celery, whereas the tomato was infected by ToMV. The genome of this secovirus was determined by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). Amplicons were cloned and Sanger sequenced with at least three independent clones per amplicon. Sequences were assembled by the SeqMan Pro 7.1.0 in the Lasergene (DNASTAR, Madison, WI). The genome of this virus is composed of two RNAs of 6,616 and 5,356 nt (excluding the polyadenylic acid tails) (GenBank accession nos. MW143070 and MW143071, respectively). The thirty contigs assembled from sRNAs could be mapped to the genome. Pairwise sequence analyses showed that RNA1 and RNA2 and their encoded polyproteins shared the highest nt (82.7% and 82.2%) and aa (93.4% and 91.8%) sequence identities with the respective RNAs (GenBank accession nos. MN412739 and MN412740) and their encoded polyproteins of snLaSV [4]. In the phylogenetic trees, this virus sequence clustered with snLaSV and LSV-1 in a separate branch neighboring viruses of the subgenus Stramovirus or Satsumavirus in the genus Sadwavirus. These results suggest that this virus is an isolate of the unclassified snLaSV and was referred as snLaSV-CHN. RT-PCR with primers SecoR1-3700F and SecoR1-5100R confirmed the presence of snLaSV-CHN in other mountain celeries (11 of 23 tested) showing chlorotic spots symptoms but not in healthy ones from the same field. To the best of our knowledge, this is the first report of snLaSV infecting mountain celery in China and a more orthodox name, mountain celery chlorotic spot virus (MCCSV), is tentatively proposed. Our findings provide a better insight of the distribution and host range of this virus and further surveys are necessary to determine its incidence and damage in mountain celery. Funding: This study is financial supported by the Program for the Scientific Activities of Selected Returned Overseas Professionals in Heilongjiang Province (Grant No. 2018QD0002) and the China National Funds for Excellent Young Scientists (Grant No. 32022071). References Son, H. J. 2020. Food Sci Nutr. 9:514. Martin, M. 2011. EMBnet J. 17:10. Che, X., et al. 2020. Plant Dis. 104: 3085. Gaafar, Y. Z. A., et al. 2020. Front Microbiol. 11: 583242.

Production of a Novel Superoxide Dismutase by Escherichia coli and Pichia pastoris and Analysis of the Thermal Stability of the Enzyme.

Previously, a new copper-zinc SOD (CuZnSOD) isolated from chestnut rose (Rosa roxburghii) with good stability was described. In this study, the biosynthetic approach was used to create recombinant CuZnSOD. RACE PCR was also used to amplify the full-length CuZnSOD gene from chestnut rose, and the ORF segment was expressed in E. coli BL21 and P. pastoris GS115. For characterization, the enzyme was isolated in two steps in E. coli and one step in P. pastoris. The biochemical properties of the two recombinant enzymes were similar, and their optimal reaction pH and temperature were 6.0 and 50°C, respectively. According to molecular dynamics simulation, the CuZnSOD showed high stability from 70 to 90°C, and eight amino acids are important for enzyme thermal stability at high temperatures. This study set the stage for industrial manufacture by filling gaps in the link between conformational changes and the thermal stability of the new CuZnSOD.

Complete genome sequence of vallota mosaic virus detected in a narcissus bulb imported from the United States to Japan.

Narcissus (Narcissus albidus) imported from the United States exhibited leaf chlorosis during post-entry quarantine. We employed next-generation sequencing (NGS) on symptomatic leaf samples and detected vallota mosaic virus (ValMV), belonging to the genus Potyvirus, family Potyviridae, as the viral agent. Sanger sequencing of PCR products and rapid amplification of cDNA ends based on NGS contigs revealed that ValMV is 9,451 nucleotides (nt) in length, excluding the poly(A) tail. Nucleotide and amino acid (aa) sequences of the coat protein region had over 98% identity to previously reported ValMV isolates. In each of the 10 regions encoding mature proteins, however, the sequence identity to other potyviruses was 49.5-71.9% nt and 18.3-78.9% aa, values that are below the species demarcation thresholds for the family Potyviridae. Phylogenetic analysis revealed that our ValMV isolate is most closely related to known ValMV isolates and is grouped with other potyviruses. Taken together, our results indicate that the newly isolated ValMV belongs to a distinct species in the genus Potyvirus. This study provides the first report of the complete ValMV genome sequence and the first record of this virus in narcissus.

Grxcr1 regulates hair bundle morphogenesis and is required for normal mechanoelectrical transduction in mouse cochlear hair cells.

Tasmanian devil (tde) mice are deaf and exhibit circling behaviour. Sensory hair cells of mutants show disorganised hair bundles with abnormally thin stereocilia. The origin of this mutation is the insertion of a transgene which disrupts expression of the Grxcr1 (glutaredoxin cysteine rich 1) gene. We report here that Grxcr1 exons and transcript sequences are not affected by the transgene insertion in tde homozygous (tde/tde) mice. Furthermore, 5’RACE PCR experiments showed the presence of two different transcripts of the Grxcr1 gene, expressed in both tde/tde and in wild-type controls. However, quantitative analysis of Grxcr1 transcripts revealed a significantly decreased mRNA level in tde/tde mice. The key stereociliary proteins ESPN, MYO7A, EPS8 and PTPRQ were distributed in hair bundles of homozygous tde mutants in a similar pattern compared with control mice. We found that the abnormal morphology of the stereociliary bundle was associated with a reduction in the size and Ca2+-sensitivity of the mechanoelectrical transducer (MET) current. We propose that GRXCR1 is key for the normal growth of the stereociliary bundle prior to the onset of hearing, and in its absence hair cells are unable to mature into fully functional sensory receptors.

A versatile 5\u2032 RACE-Seq methodology for the accurate identification of the 5\u2032 termini of mRNAs

BackgroundTechnological advancements in the era of massive parallel sequencing have enabled the functional dissection of the human transcriptome. However, 5′ ends of mRNAs are significantly underrepresented in these datasets, hindering the efficient analysis of the complex human transcriptome. The implementation of the template-switching mechanism at the reverse transcription stage along with 5′ rapid amplification of cDNA ends (RACE) constitutes the most prominent and efficient strategy to specify the actual 5′ ends of cDNAs. In the current study, we developed a 5′ RACE-seq method by coupling a custom template-switching and 5′ RACE assay with targeted nanopore sequencing, to accurately unveil 5′ termini of mRNA targets.ResultsThe optimization of the described 5′ RACE-seq method was accomplished using the human BCL2L12 as control gene. We unveiled that the selection of hybrid DNA/RNA template-switching oligonucleotides as well as the complete separation of the cDNA extension incubation from the template-switching process, significantly increase the overall efficiency of the downstream 5′ RACE. Collectively, our results support the existence of two distinct 5′ termini for BCL2L12, being in complete accordance with the results derived from both direct RNA and PCR-cDNA sequencing approaches from Oxford Nanopore Technologies. As proof of concept, we implemented the described 5′ RACE-seq methodology to investigate the 5′ UTRs of several kallikrein-related peptidases (KLKs) gene family members. Our results confirmed the existence of multiple annotated 5′ UTRs of the human KLK gene family members, but also identified novel, previously uncharacterized ones.ConclusionsIn this work we present an in-house developed 5′ RACE-seq method, based on the template-switching mechanism and targeted nanopore sequencing. This approach enables the broad and in-depth study of 5′ UTRs of any mRNA of interest, by offering a tremendous sequencing depth, while significantly reducing the cost-per reaction compared to commercially available kits.

Genome Characterization and Phylogenetic Analysis of a Novel Endornavirus That Infects Fungal Pathogen Sclerotinia sclerotiorum.

Endornaviruses are capsidless linear (+) ssRNA viruses in the family Endornaviridae. In this study, Scelrotinia sclerotiorum endornavirus 11 (SsEV11), a novel endornavirus infecting hypovirulent Sclerotinia sclerotiorum strain XY79, was identified and cloned using virome sequencing analysis and rapid amplification of cDNA ends (RACE) techniques. The full-length genome of SsEV11 is 11906 nt in length with a large ORF, which encodes a large polyprotein of 3928 amino acid residues, containing a viral methyltransferase domain, a cysteine-rich region, a putative DEADc, a viral helicase domain, and an RNA-dependent RNA polymerase (RdRp) 2 domain. The 5’ and 3’ untranslated regions (UTR) are 31 nt and 90 nt, respectively. According to the BLAST result of the nucleotide sequence, SsEV11 shows the highest identity (45%) with Sclerotinia minor endornavirus 1 (SmEV1). Phylogenetic analysis based on amino acid sequence of RdRp demonstrated that SsEV11 clusters to endornavirus and has a close relationship with Betaendornavirus. Phylogenetic analysis based on the sequence of endornaviral RdRp domain indicated that there were three large clusters in the phylogenetic tree. Combining the results of alignment analysis, Cluster I at least has five subclusters including typical members of Alphaendornavirus and many unclassified endornaviruses that isolated from fungi, oomycetes, algae, and insects; Cluster II also has five subclusters including typical members of Betaendornavirus, SsEV11, and other unclassified viruses that infected fungi; Cluster III includes many endorna-like viruses that infect nematodes, mites, and insects. Viruses in Cluster I and Cluster II are close to each other and relatively distant to those in Cluster III. Our study characterized a novel betaendornavirus, SsEV11, infected fungal pathogen S. sclerotiorum, and suggested that notable phylogenetic diverse exists in endornaviruses. In addition, at least, one novel genus, Gammaendornavirus, should be established to accommodate those endorna-like viruses in Cluster III.