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mutational clone

Chaowen Zhang, Feifan Chen, Ziyao Zhao, Liangliang Hu, Hanqiang Liu, Zhihui Cheng, Yiqun Weng, Peng Chen, Yuhong Li
Two round-leaf mutants, rl-1 and rl-2, were identified from EMS-induced mutagenesis. High throughput sequencing and map-based cloning suggested CsPID encoding a Ser/Thr protein kinase as the most possible candidate for rl-1. Rl-2 was allelic to Rl-1. Leaf shape is an important plant architecture trait that is affected by plant hormones, especially auxin. In Arabidopsis, PINOID (PID), a regulator for the auxin polar transporter PIN (PIN-FORMED) affects leaf shape formation, but this function of PID in crop plants has not been well studied...
March 14, 2018: TAG. Theoretical and Applied Genetics. Theoretische und Angewandte Genetik
Christian E Ogaugwu, Qiuying Cheng, Annabeth Fieck, Ivy Hurwitz, Ravi Durvasula
Constitutively active promoter elements for heterologous protein production in Lactococcus lactis are scarce. Here, the promoter of the PTS-IIC gene cluster from L. lactis NZ3900 is described. This promoter was cloned upstream of an enhanced green fluorescent protein, GFPmut3a, and transformed into L. lactis. Transformants produced up to 13.5 μg of GFPmut3a per milliliter of log phase cells. Addition of cellobiose further increased the production of GFPmut3a by up to two-fold when compared to glucose. Analysis of mutations at two specific positions in the PTS-IIC promoter showed that a 'T' to 'G' mutation within the -35 element resulted in constitutive expression in glucose, while a 'C' at nucleotide 7 in the putative cre site enhanced promoter activity in cellobiose...
March 2018: Biotechnology Reports
Taigo Kato, Tatsuo Matsuda, Yuji Ikeda, Jae-Hyun Park, Matthias Leisegang, Sachiko Yoshimura, Tetsuro Hikichi, Makiko Harada, Makda Zewde, Sho Sato, Kosei Hasegawa, Kazuma Kiyotani, Yusuke Nakamura
Neoantigens are the main targets of tumor-specific T cells reactivated by immune checkpoint-blocking antibodies or when using tumor-infiltrating T cells for adoptive therapy. While cancers often accumulate hundreds of mutations and harbor several immunogenic neoantigens, the repertoire of mutation-specific T cells in patients might be restricted. To bypass suboptimal conditions, which impede the reactivation of existing T cells or the priming of neoantigen-specific T cells in a patient, we employ T cells of healthy donors with an overlapping HLA repertoire to target cancer neoantigens...
February 16, 2018: Oncotarget
Yannan Zhao, Lilan Luo, Jiesi Xu, Peiyong Xin, Hongyan Guo, Jian Wu, Lin Bai, Guodong Wang, Jinfang Chu, Jianru Zuo, Hong Yu, Xun Huang, Jiayang Li
Programmed cell death (PCD) is a fundamental biological process. Deficiency in MOSAIC DEATH 1 (MOD1), a plastid-localized enoyl-ACP reductase, leads to the accumulation of reactive oxygen species (ROS) and PCD, which can be suppressed by mitochondrial complex I mutations, indicating a signal from chloroplasts to mitochondria. However, this signal remains to be elucidated. In this study, through cloning and analyzing a series of mod1 suppressors, we reveal a comprehensive organelle communication pathway that regulates the generation of mitochondrial ROS and triggers PCD...
March 14, 2018: Cell Research
Alan D Marmorstein, Adiv A Johnson, Lori A Bachman, Cynthia Andrews-Pfannkoch, Travis Knudsen, Benjamin J Gilles, Matthew Hill, Jarel K Gandhi, Lihua Y Marmorstein, Jose S Pulido
Autosomal recessive bestrophinopathy (ARB) is caused by mutations in the gene BEST1 which encodes bestrophin 1 (Best1), an anion channel expressed in retinal pigment epithelial (RPE) cells. It has been hypothesized that ARB represents the human null phenotype for BEST1 and that this occurs due to nonsense mediated decay (NMD). To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with ARB and her parents. After differentiation to retinal pigment epithelial (iPSC-RPE) cells, both BEST1 mRNA and Best1 protein expression were compared to controls...
March 14, 2018: Scientific Reports
Jin Yang Baek, Cheol-In Kang, So Hyun Kim, Kwan Soo Ko, Doo Ryeon Chung, Kyong Ran Peck, Nam Yong Lee, Jae-Hoon Song
The use of fluoroquinolones to treat respiratory tract infections and pneumonia due to Streptococcus pneumoniae has affected the emergence of resistance to this class of drugs. Increasing pneumococcal resistance to levofloxacin has become a major public health concern. We investigated the prevalence and genetic characteristics of levofloxacin-nonsusceptible S. pneumoniae (LNSP) clinical isolates in Korea. A total of 43 LNSP isolates collected from a national surveillance study at 13 tertiary hospitals between 2008 and 2014 were analyzed for serotype and antimicrobial susceptibilities to 19 antimicrobial agents as well as the quinolone resistance-determining region mutation...
February 16, 2018: Diagnostic Microbiology and Infectious Disease
Frances Rocamora, Lei Zhu, Kek Yee Liong, Arjen Dondorp, Olivo Miotto, Sachel Mok, Zbynek Bozdech
Due to their remarkable parasitocidal activity, artemisinins represent the key components of first-line therapies against Plasmodium falciparum malaria. However, the decline in efficacy of artemisinin-based drugs jeopardizes global efforts to control and ultimately eradicate the disease. To better understand the resistance phenotype, artemisinin-resistant parasite lines were derived from two clones of the 3D7 strain of P. falciparum using a selection regimen that mimics how parasites interact with the drug within patients...
March 14, 2018: PLoS Pathogens
Hernan Garcia-Ruiz, Sergio M Gabriel Peralta, Patricia A Harte-Maxwell
Plant viruses are inducers and targets of antiviral RNA silencing. To condition susceptibility, most plant viruses encode silencing suppressor proteins that interfere with antiviral RNA silencing. The NSs protein is an RNA silencing suppressor in orthotospoviruses, such as the tomato spotted wilt virus (TSWV). The mechanism of RNA silencing suppression by NSs and its role in virus infection and movement are poorly understood. Here, we cloned and tagged TSWV NSs and expressed it from a GFP-tagged turnip mosaic virus (TuMV-GFP) carrying either a wild-type or suppressor-deficient (AS9) helper component proteinase (HC-Pro)...
March 14, 2018: Viruses
Mengzhu Li, Hong Deng, Rui Ma, Huiying Luo, Bin Yao, Xiaoyun Su
Pyranose oxidase (POx) is a homotetrameric flavoprotein that catalyzes the oxidation of pyranose-configured sugars at position C-2 to corresponding 2-ketoaldoses. The wide substrate specificity makes POx potential for application in various biotechnological industries. In the present study we reported the gene cloning and heterologous expression of a POx from the basidiomycete Trametes sp. and functionally expressed the gene in Escherichia coli BL21(DE3). Based on sequence alignment, three residues were chosen for site-directed mutagenesis to obtain two single mutants (K312E and E539K) and two double mutants (T166A/E539K and K312E/E539K)...
March 13, 2018: AMB Express
Takahiro Nakagomi, Taichiro Goto, Yosuke Hirotsu, Daichi Shikata, Yujiro Yokoyama, Rumi Higuchi, Kenji Amemiya, Kenichiro Okimoto, Toshio Oyama, Hitoshi Mochizuki, Masao Omata
Objectives: Pulmonary sarcomatoid carcinomas are rare and generally aggressive tumors composed of carcinomatous and sarcomatous components; however, the evolution of sarcomatoid cancer has not been elucidated. Here, we aimed to evaluate the mutational profiles and phylogeny of sarcomatoid carcinomas using next generation sequencing and in-silico analysis to facilitate the development of novel therapies. Methods: Four patients who underwent surgery for sarcomatoid cancer were enrolled...
February 13, 2018: Oncotarget
Kinh Van Nguyen, Trung Vu Nguyen, Hang Thi Thuy Nguyen, Duyet Van Le
Introduction: Pseudomonas aeruginosa has many mechanisms of resistance to fluoroquinolones. The main mechanism is to change the effect of two enzymes that open the DNA helix - the enzyme DNA gyrase ( gyrA ) and the topoisomerase IV ( parC ). In addition, mutations that render the MexAB-oprM pump ( mexR ) dysfunctional, leading to its overexpression, also enhance resistance to fluoroquinolones. In this study, we aim to detect point mutations of gyrA , parC , and mexR genes that are predicted to be associated with fluoroquinolone resistance in 141 fluoroquinolone-resistant clinical isolates of P...
2018: Infection and Drug Resistance
Ke Xiong, Suyue Xiong, Siyu Gao, Qin Li, Baoguo Sun, Xiuting Li
The preparation of oligosaccharides via xylan hydrolysis is an effective way to add value to hemicellulosic material of agricultural waste. The bacterial strain Streptomyces L10608, isolated from soil, contains genes encoding xylanases of glucoside hydrolase family 10/11 (GH10/11), and these have been cloned to catalyze the production of xylooligosaccharide (XOS). To improve the XOS proportion of hydrolysates produced by xylanase, four amino acid residues were substituted by site-directed mutagenesis, and the mutant genes were overexpressed in Escherichia coli ...
March 13, 2018: International Journal of Molecular Sciences
Javier Viña-Gonzalez, Katarina Elbl, Xavier Ponte, Francisco Valero, Miguel Alcalde
Aryl-alcohol oxidase (AAO) plays a fundamental role in the fungal ligninolytic secretome, acting as a supplier of H2 O2 . Despite its highly selective mechanism of action, the presence of this flavooxidase in different biotechnological settings has hitherto been hampered by the lack of appropriate heterologous expression systems. We recently described the functional expression of the AAO from Pleurotus eryngii in Saccharomyces cerevisiae by fusing a chimeric signal peptide (preαproK) and applying structure-guided evolution...
March 13, 2018: Biotechnology and Bioengineering
Zhi-Jie Wu, Xin Zhao, Lauren G Banaszak, Fernanda Gutierrez-Rodrigues, Keyvan Keyvanfar, Shou-Guo Gao, Diego Quinones Raffo, Sachiko Kajigaya, Neal S Young
Additional sex combs-like 1 (ASXL1) is a well‑known tumor suppressor gene and epigenetic modifier. ASXL1 mutations are frequent in myeloid malignances; these mutations are risk factors for the development of myelodysplasia and also appear as small clones during normal aging. ASXL1 appears to act as an epigenetic regulator of cell survival and myeloid differentiation; however, the molecular mechanisms underlying the malignant transformation of cells with ASXL1 mutations are not well defined. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing, heterozygous and homozygous ASXL1 mutations were introduced into human U937 leukemic cells...
April 2018: International Journal of Oncology
Sri I Pulungan, Ryoichi Yano, Yoshihiro Okabe, Takuji Ichino, Mikiko Kojima, Yumiko Takebayashi, Hitoshi Sakakibara, Tohru Ariizumi, Hiroshi Ezura
Leaves are the major plant organs with a primary function for photosynthesis. Auxin controls various aspects of plant growth and development, including leaf initiation, expansion, and differentiation. Unique and intriguing auxin features include its polar transport, which is mainly controlled by the AUX1/LAX and PIN gene families as influx and efflux carriers, respectively. The role of AUX1/ LAX genes in root development is well documented, but the role of these genes in leaf morphogenesis remains unclear. Moreover, most studies have been conducted in the plant model Arabidopsis thaliana, while studies in tomato are still scarce...
March 8, 2018: Plant & Cell Physiology
Younguk Sun, Bo-Rui Chen, Aniruddha Deshpande
The importance of epigenetic dysregulation to acute myeloid leukemia (AML) pathophysiology has become increasingly apparent in recent years. Epigenetic regulators, including readers, writers, and erasers, are recurrently dysregulated by way of chromosomal translocations, somatic mutations, or genomic amplification in AML and many of these alterations are directly implicated in AML pathogenesis. Mutations in epigenetic regulators are often discovered in founder clones and persist after therapy, indicating that they may contribute to a premalignant state poised for the acquisition of cooperating mutations and frank malignancy...
2018: Frontiers in Oncology
Helen Fifer, Michelle Cole, Gwenda Hughes, Simon Padfield, Christa Smolarchuk, Neil Woodford, Adrian Wensley, Nazim Mustafa, Ulf Schaefer, Richard Myers, Kate Templeton, Jill Shepherd, Anthony Underwood
BACKGROUND: Between Nov 3, 2014, and Feb 24, 2017, 70 cases of high-level azithromycin-resistant (HL-AziR; minimum inhibitory concentration [MIC] ≥256 mg/L) Neisseria gonorrhoeae were reported from across England. Whole-genome sequencing was done to investigate this outbreak to determine whether the ongoing outbreak represented clonal spread of an HL-AziR N gonorrhoeae strain identified in Leeds. We also wanted to elucidate the molecular mechanisms of azithromycin resistance in N gonorrhoeae in the UK...
March 6, 2018: Lancet Infectious Diseases
Fulvio Massaro, Gioia Colafigli, Matteo Molica, Massimo Breccia
Chronic myeloid leukemia (CML) is characterized by a pathognomonic chromosomal translocation, which leads to the fusion of breakpoint cluster region (BCR) and Abelson leukemia virus 1 (ABL1) genes, generating an oncoprotein with deregulated tyrosine kinase activity. Areas covered: In the last two decades, BCR/ABL1 kinase has become the molecular target for tyrosine kinase inhibitors (TKIs), a class of drugs that impressively improved overall survival. Despite these results, a proportion of patients experiences resistance to TKIs and need to change treatment...
March 9, 2018: Expert Review of Hematology
Qiuju Chen, Dong Meng, Zhaoyu Gu, Wei Li, Hui Yuan, Xuwei Duan, Qing Yang, Yang Li, Tianzhong Li
It has been proved that the gametophytic self-incompatibility (GSI), mainly exists in Rosaceae and Solanaceae, is controlled by S genes, which are two tightly linked genes located at highly polymorphic S -locus: the S-RNase for pistil specificity and the F-box gene ( SFB/SLF ) for pollen specificity, respectively. However, the roles of those genes in SI of peach are still a subject of extensive debate. In our study, we selected 37 representative varieties according to the evolution route of peach and identified their S genotypes...
2018: Frontiers in Plant Science
Moritz Horn, Virginia Kroef, Kira Allmeroth, Nicole Schuller, Stephan Miethe, Martin Peifer, Josef M Penninger, Ulrich Elling, Martin S Denzel
Forward genetic screens in haploid mammalian cells have recently emerged as powerful tools for the discovery and investigation of recessive traits. Use of the haploid system provides unique genetic tractability and resolution. Upon positive selection, these screens typically employ analysis of loss-of-function (LOF) alleles and are thus limited to non-essential genes. Many relevant compounds, including anti-cancer therapeutics, however, target essential genes, precluding positive selection of LOF alleles. Here, we asked whether the use of random and saturating chemical mutagenesis might enable screens that identify essential biological targets of toxic compounds...
February 9, 2018: Oncotarget
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