• Mycobiology
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• v.50 no.4
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• pp.254-257
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• 2022

Wolfiporia cocos is a wood-decay brown rot fungus belonging to the family Polyporaceae. While the fungus grows, the sclerotium body of the strain, dubbed Bokryeong in Korean, is formed around the roots of conifer trees. The dried sclerotium has been widely used as a key component of many medicinal recipes in East Asia. Wolfiporia cocos strain KMCC03342 is the reference strain registered and maintained by the Korea Seed and Variety Service for commercial uses. Here, we present the first draft genome sequence of W. cocos KMCC03342 using a hybrid assembly technique combining both short- and long-read sequences. The genome has a total length of 55.5 Mb comprised of 343 contigs with N50 of 332 kb and 95.8% BUSCO completeness. The GC ratio was 52.2%. We predicted 14,296 protein-coding gene models based on ab initio gene prediction and evidence-based annotation procedure using RNAseq data. The annotated genome was predicted to have 19 terpene biosynthesis gene clusters, which was the same number as the previously sequenced W. cocos strain MD-104 genome but higher than Chinese W. cocos strains. The genome sequence and the predicted gene clusters allow us to study biosynthetic pathways for the active ingredients of W. cocos.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.714-718
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• 1998

Various mutants either lacking or having decreased levels of light-harvesting complexes and reaction center complex were obtained with a high frequency by an increased formation of poly-3-hydroxybutyrate (PHB) in Rhodobacter sphaeroides. One of the photosynthesis-defective mutants, PY-17, which was devoid of any of the light-harvesting complexes (B800-850, B875) as well as the reaction center complex, was analyzed further. The mutant showed substantial transcription of the puhA, pufKBALMX, and pucBAC operons coding for the structural proteins of the photosynthetic complexes although each of the activities was lower than that of the wild type. Translation of the pufKBALMX and pucBAC operons were also active in the mutant although with activities different from the corresponding one of the wild type. From these results the mutation appears to exert its effect at the post-translational level of the photosynthetic complex assembly. Complementation of the photosynthesis-defective phenotype of the mutant was achieved with an about 12-kb DNA region containing the puhA gene. The relationship between the formation of PHB and photosynthetic complexes is discussed.

• Molecules and Cells
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• v.41 no.4
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• pp.301-310
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• 2018

LysR-type transcriptional regulators (LTTRs) contain an N-terminal DNA binding domain (DBD) and a C-terminal regulatory domain (RD). Typically, LTTRs function as homotetramers. VV2_1132 was identified in Vibrio vulnificus as an LTTR that is a homologue of HypT (also known as YjiE or QseD) in Escherichia coli. In this study, we determined the crystal structure of full-length VV2_1132 at a resolution of $2.2{\AA}$, thereby revealing a novel combination of the domains in the tetrameric assembly. Only one DBD dimer in the tetramer can bind to DNA, because the DNA binding motifs of the other DBD dimer are completely buried in the tetrameric assembly. Structural and functional analyses of VV2_1132 suggest that it might not perform the same role as E. coli HypT, indicating that further study is required to elucidate the function of this gene in V. vulnificus. The unique structure of VV2_1132 extends our knowledge of LTTR function and mechanisms of action.

• BMB Reports
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• v.41 no.12
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• pp.868-874
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• 2008

Neurofilaments (NFs) are neuronal intermediate filaments composed of light (NF-L), middle (NF-M), and heavy (NF-H) subunits. NF-L self-assembles into a "core" filament with which NF-M or NF-H co-assembles to form the neuronal intermediate filament. Recent reports show that point mutations of the NF-L gene result in Charcot-Marie-Tooth disease (CMT). However, the most recently described rod domain mutant of human NF-L (A148V) has not been characterized in cellular level. We cloned human NF-L and used it to engineer the A148V. In phenotypic analysis using SW13 cells, A148V mutation completely abolished filament formation despite of presence of NF-M. Moreover, A148V mutation reduced the levels of in vitro self-assembly using GST-NF-L (H/R) fusion protein whereas control (A296T) mutant did not affect the filament formation. These results suggest that alanine at position 148 is essentially required for NF-L self-assembly leading to subsequent filament formation in neuronal cells.

• Genomics & Informatics
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• v.10 no.1
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• pp.1-8
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• 2012

Recently, the technologies of DNA sequence variation and gene expression profiling have been used widely as approaches in the expertise of genome biology and genetics. The application to genome study has been particularly developed with the introduction of the nextgeneration DNA sequencer (NGS) Roche/454 and Illumina/ Solexa systems, along with bioinformation analysis technologies of whole-genome $de$ $novo$ assembly, expression profiling, DNA variation discovery, and genotyping. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing $de$ $novo$ assembly of novel genome sequencing data. It is necessary to have DNA sequence information from a multiplatform NGS with at least $2{\times}$ and $30{\times}$ depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly. Massive shortlength reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a wholegenome transcriptome, depending on the tissue samples. The hybrid mRNA sequences from Rohce/454 and Illumina/Solexa are more powerful to find novel genes through $de$ $novo$ assembly in any whole-genome sequenced species. The $20{\times}$ and $50{\times}$ coverage of the estimated transcriptome sequences using Roche/454 and Illumina/Solexa, respectively, is effective to create novel expressed reference sequences. However, only an average $30{\times}$ coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference expressed sequence tag sequence.

• Fisheries and Aquatic Sciences
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• v.10 no.3
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• pp.119-126
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• 2007

The analysis of expressed sequence tags (ESTs) is an efficient approach for gene discovery, expression profiling, and the development of resources for functional genomics. To analyze the transcriptome of the abalone Haliotis discus hannai, we conducted EST analysis using seven cDNA libraries made from gill, gut, hepatopancreas, skin, muscle, testis, and ovary. Redundant ESTs were assembled into overlapping contiguous sequences using the assembly program ICAtools. We found that the total 1,393 ESTs formed 135 clusters and 951 singletons, indicating that the overall redundancy of the library was 22%. Of the 1,393 clones, BLAST identified 1,278 clones (91.7%) as known genes; 115 clones (8.3%) did not match any previously described gene. Based on the major functions of their encoded proteins, the identified clones were classified into 16 broad categories. Sequence analysis revealed the presence of micro satellite-containing genes that may be valuable for further gene mapping studies. This study contributes to the identification of numerous EST clones that can be applied to further clarifying the genetics and developmental biology of abalone.

• Microbiology and Biotechnology Letters
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• v.49 no.3
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• pp.305-315
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• 2021

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.

• Genomics & Informatics
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• v.19 no.1
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• pp.8.1-8.12
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• 2021

Cytorace-3 is a laboratory evolved hybrid lineage of Drosophila nasuta nasuta males and Drosophila nasuta albomicans females currently passing ~850 generations. To assess interracial hybridization effects on gene expression in Cytorace-3 we profiled the transcriptomes of mature ovaries and testes by employing Illumina sequencing technology and de novo transcriptome assembling strategies. We found 26% of the ovarian, and 14% of testis genes to be differentially expressed in Cytorace-3 relative to the expressed genes in the parental gonadal transcriptomes. About 5% of genes exhibited additive gene expression pattern in the ovary and 3% in the testis, while the remaining genes were misexpressed in Cytorace-3. Nearly 772 of these misexpressed genes in the ovary and 413 in the testis were either over-or under-dominant. Genes following D. n. nasuta dominance was twice (270 genes) than D. n. albomicans dominance (133 genes) in the ovary. In contrast, only 105 genes showed D. n. nasuta dominance and 207 showed D. n. albomicans dominance in testis transcriptome. Of the six expression inheritance patterns, conserved inheritance pattern was predominant for both ovary (73%) and testis (85%) in Cytorace-3. This study is the first to provide an overview of the expression divergence and inheritance patterns of the transcriptomes in an independently evolving distinct hybrid lineage of Drosophila. This recorded expression divergence in Cytorace-3 surpasses that between parental lineages illustrating the strong impact of hybridization driving rapid gene expression changes.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.8-15
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• 2021

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

• The Plant Pathology Journal
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• v.37 no.6
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• pp.619-631
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• 2021

Alfalfa mosaic virus (AMV), an economically important pathogen, is present worldwide with a very wide host range. This work reports for the first time the infection of Vinca minor and Wisteria sinensis with AMV using RNA sequencing and reverse transcription polymerase chain reaction confirmation. De novo assembly and annotating of contigs revealed that RNA1, RNA2, and RNA3 genomic fragments consist of 3,690, 2,636, and 2,057 nucleotides (nt) for IR-VM and 3,690, 2,594, and 2,057 nt for IR-WS. RNA1 and RNA3 segments of IR-VM and IR-WS closely resembled those of the Chinese isolate HZ, with 99.23-99.26% and 98.04-98.09% nt identity, respectively. Their RNA2 resembled that of Canadian isolate CaM and American isolate OH-2-2017, with 97.96-98.07% nt identity. The P2 gene revealed more nucleotide diversity compared with other genes. Genes in the AMV genome were under dominant negative selection during evolution, and the P1 and coat protein (CP) proteins were subject to the strongest and weakest purifying selection, respectively. In the population genetic analysis based on the CP gene sequences, all 107 AMV isolates fell into two main clades (A, B) and isolates of clade A were further divided into three groups with significant subpopulation differentiation. The results indicated moderate genetic variation within and no clear geographic or genetic structure between the studied populations, implying moderate gene flow can play an important role in differentiation and distribution of genetic diversity among populations. Several factors have shaped the genetic structure and diversity of AMV: selection, recombination/reassortment, gene flow, and random processes such as founder effects.