scholarly journals Searching for a Needle in a Haystack: Cas9-Targeted Nanopore Sequencing and DNA Methylation Profiling of Full-Length Glutenin Genes in a Big Cereal Genome

Plants ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 5
Author(s):  
Ilya Kirov ◽  
Ekaterina Polkhovskaya ◽  
Maxim Dudnikov ◽  
Pavel Merkulov ◽  
Anastasia Vlasova ◽  
...  
Keyword(s):  
Genome Size ◽  
Target Gene ◽  
Target Genes ◽  
Gene Expression Regulation ◽  
Full Length ◽  
Nanopore Sequencing ◽  
Gene Body ◽  
Gene Body Methylation ◽  
Marker Design ◽  
Methylation Profiling

Sequencing and epigenetic profiling of target genes in plants are important tasks with various applications ranging from marker design for plant breeding to the study of gene expression regulation. This is particularly interesting for plants with big genome size for which whole-genome sequencing can be time-consuming and costly. In this study, we asked whether recently proposed Cas9-targeted nanopore sequencing (nCATS) is efficient for target gene sequencing for plant species with big genome size. We applied nCATS to sequence the full-length glutenin genes (Glu-1Ax, Glu-1Bx and Glu-1By) and their promoters in hexaploid triticale (X Triticosecale, AABBRR, genome size is 24 Gb). We showed that while the target gene enrichment per se was quite high for the three glutenin genes (up to 645×), the sequencing depth that was achieved from two MinION flowcells was relatively low (5–17×). However, this sequencing depth was sufficient for various tasks including detection of InDels and single-nucleotide variations (SNPs), read phasing and methylation profiling. Using nCATS, we uncovered SNP and InDel variation of full-length glutenin genes providing useful information for marker design and deciphering of variation of individual Glu-1By alleles. Moreover, we demonstrated that glutenin genes possess a ‘gene-body’ methylation epigenetic profile with hypermethylated CDS part and hypomethylated promoter region. The obtained information raised an interesting question on the role of gene-body methylation in glutenin gene expression regulation. Taken together, our work disclosures the potential of the nCATS approach for sequencing of target genes in plants with big genome size.

Artificial microRNA (amiRNA) induced gene silencing in alfalfa (Medicago sativa)

Botany ◽  
2013 ◽  
Vol 91 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Julian C. Verdonk ◽  
Michael L. Sullivan
Keyword(s):  
Gene Silencing ◽  
Medicago Sativa ◽  
Target Gene ◽  
Target Genes ◽  
Mirna Precursor ◽  
Crop Species ◽  
Forage Crop ◽  
Ribonucleic Acids ◽  
Endogenous Gene ◽  
Medicago Sativa L

Gene silencing is a powerful technique that allows the study of the function of specific genes by selectively reducing their transcription. Several different approaches can be used, however they all have in common the artificial generation of single stranded small ribonucleic acids (RNAs) that are utilized by the endogenous gene silencing machinery of the organism. Artificial microRNAs (amiRNA) can be used to very specifically target genes for silencing because only a short sequence of 21 nucleotides of the gene of interest is used. Gene silencing via amiRNA has been developed for Arabidopsis thaliana (L.) Heynh. and rice using endogenous microRNA (miRNA) precursors and has been shown to also work effectively in other dicot species using the arabidopsis miRNA precursor. Here, we demonstrate that the arabidopsis miR319 precursor can be used to silence genes in the important forage crop species alfalfa (Medicago sativa L.) by silencing the expression of a transgenic beta-glucuronidase (GUSPlus) target gene.

scholarly journals CHOP-Dependent Stress-Inducible Expression of a Novel Form of Carbonic Anhydrase VI

1999 ◽  
Vol 19 (1) ◽  
pp. 495-504 ◽  
Author(s):  
John Sok ◽  
Xiao-Zhong Wang ◽  
Nikoleta Batchvarova ◽  
Masahiko Kuroda ◽  
Heather Harding ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Target Gene ◽  
Target Genes ◽  
Direct Evidence ◽  
Nuclear Protein ◽  
Intracellular Form ◽  
Carbonic Anhydrase Vi ◽  
Responsive Element

ABSTRACT CHOP (also called GADD153) is a stress-inducible nuclear protein that dimerizes with members of the C/EBP family of transcription factors and was initially identified as an inhibitor of C/EBP binding to classic C/EBP target genes. Subsequent experiments suggested a role for CHOP-C/EBP heterodimers in positively regulating gene expression; however, direct evidence that this is the case has so far not been uncovered. Here we describe the identification of a positively regulated direct CHOP-C/EBP target gene, that encoding murine carbonic anhydrase VI (CA-VI). The stress-inducible form of the gene is expressed from an internal promoter and encodes a novel intracellular form of what is normally a secreted protein. Stress-induced expression of CA-VI is both CHOP and C/EBPβ dependent in that it does not occur in cells deficient in either gene. A CHOP-responsive element was mapped to the inducibleCA-VI promoter, and in vitro footprinting revealed binding of CHOP-C/EBP heterodimers to that site. Rescue of CA-VIexpression in c/ebpβ−/− cells by exogenous C/EBPβ and a shorter, normally inhibitory isoform of the protein known as LIP suggests that the role of the C/EBP partner is limited to targeting the CHOP-containing heterodimer to the response element and points to a preeminent role for CHOP in CA-VI induction during stress.

scholarly journals Mutant Alleles of the Drosophila trithorax Gene Produce Common and Unusual Homeotic and Other Developmental Phenotypes

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 319-344
Author(s):  
Thomas R Breen
Keyword(s):  
Target Gene ◽  
Target Genes ◽  
Protein Isoforms ◽  
Homeotic Genes ◽  
Set Domain ◽  
Human Homologue ◽  
Terminal Set ◽  
Hypomorphic Alleles ◽  
Different Levels ◽  
Target Gene Transcription

Abstract trithorax (trx) encodes chromosome-binding proteins required throughout embryogenesis and imaginal development for tissue- and cell-specific levels of transcription of many genes including homeotic genes of the ANT-C and BX-C. trx encodes two protein isoforms that contain conserved motifs including a C-terminal SET domain, central PHD fingers, an N-terminal DNA-binding homology, and two short motifs also found in the TRX human homologue, ALL1. As a first step to characterizing specific developmental functions of TRX, I examined phenotypes of 420 combinations of 21 trx alleles. Among these are 8 hypomorphic alleles that are sufficient for embryogenesis but provide different levels of trx function at homeotic genes in imaginal cells. One allele alters the N terminus of TRX, which severely impairs larval and imaginal growth. Hypomorphic alleles that alter different regions of TRX equivalently reduce function at affected genes, suggesting TRX interacts with common factors at different target genes. All hypomorphic alleles examined complement one another, suggesting cooperative TRX function at target genes. Comparative effects of hypomorphic genotypes support previous findings that TRX has tissue-specific interactions with other factors at each target gene. Some hypomorphic genotypes also produce phenotypes that suggest TRX may be a component of signal transduction pathways that provide tissue- and cell-specific levels of target gene transcription.

scholarly journals Transcriptomic and epigenomic remodeling occurs during vascular cambium periodicity in Populus tomentosa

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Bo Chen ◽  
Huimin Xu ◽  
Yayu Guo ◽  
Paul Grünhofer ◽  
Lukas Schreiber ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Plant Hormone ◽  
Target Genes ◽  
Populus Tomentosa ◽  
Differentially Expressed ◽  
Vascular Cambium ◽  
Differentially Methylated Region ◽  
Gene Body Methylation ◽  
Transcription Analysis ◽  
Global Methylation

AbstractTrees in temperate regions exhibit evident seasonal patterns, which play vital roles in their growth and development. The activity of cambial stem cells is the basis for regulating the quantity and quality of wood, which has received considerable attention. However, the underlying mechanisms of these processes have not been fully elucidated. Here we performed a comprehensive analysis of morphological observations, transcriptome profiles, the DNA methylome, and miRNAs of the cambium in Populus tomentosa during the transition from dormancy to activation. Anatomical analysis showed that the active cambial zone exhibited a significant increase in the width and number of cell layers compared with those of the dormant and reactivating cambium. Furthermore, we found that differentially expressed genes associated with vascular development were mainly involved in plant hormone signal transduction, cell division and expansion, and cell wall biosynthesis. In addition, we identified 235 known miRNAs and 125 novel miRNAs. Differentially expressed miRNAs and target genes showed stronger negative correlations than other miRNA/target pairs. Moreover, global methylation and transcription analysis revealed that CG gene body methylation was positively correlated with gene expression, whereas CHG exhibited the opposite trend in the downstream region. Most importantly, we observed that the number of CHH differentially methylated region (DMR) changes was the greatest during cambium periodicity. Intriguingly, the genes with hypomethylated CHH DMRs in the promoter were involved in plant hormone signal transduction, phenylpropanoid biosynthesis, and plant–pathogen interactions during vascular cambium development. These findings improve our systems-level understanding of the epigenomic diversity that exists in the annual growth cycle of trees.

scholarly journals The EAR Motif in the Arabidopsis MADS Transcription Factor AGAMOUS-Like 15 Is Not Necessary to Promote Somatic Embryogenesis

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 758
Author(s):  
Sanjay Joshi ◽  
Christian Keller ◽  
Sharyn E. Perry
Keyword(s):  
Gene Expression ◽  
Somatic Embryogenesis ◽  
Target Gene ◽  
Target Genes ◽  
Domain Family ◽  
Binding Factor ◽  
Ear Motif ◽  
Carboxyl Terminal ◽  
Responsive Element ◽  
Carboxy Terminal

AGAMOUS-like 15 (AGL15) is a member of the MADS domain family of transcription factors (TFs) that can directly induce and repress target gene expression, and for which promotion of somatic embryogenesis (SE) is positively correlated with accumulation. An ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif of form LxLxL within the carboxyl-terminal domain of AGL15 was shown to be involved in repression of gene expression. Here, we examine whether AGL15′s ability to repress gene expression is needed to promote SE. While a form of AGL15 where the LxLxL is changed to AxAxA can still promote SE, another form with a strong transcriptional activator at the carboxy-terminal end, does not promote SE and, in fact, is detrimental to SE development. Select target genes were examined for response to the different forms of AGL15.

scholarly journals On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

2021 ◽  
Author(s):  
Philipp Moritz Fricke ◽  
Angelika Klemm ◽  
Michael Bott ◽  
Tino Polen
Keyword(s):  
Gene Expression ◽  
Acetic Acid ◽  
Literature Search ◽  
Target Gene ◽  
Target Genes ◽  
Recombinant Dna ◽  
Acetic Acid Bacteria ◽  
Homoserine Lactone ◽  
Expression Systems ◽  
Target Gene Expression

Abstract Acetic acid bacteria (AAB) are valuable biocatalysts for which there is growing interest in understanding their basics including physiology and biochemistry. This is accompanied by growing demands for metabolic engineering of AAB to take advantage of their properties and to improve their biomanufacturing efficiencies. Controlled expression of target genes is key to fundamental and applied microbiological research. In order to get an overview of expression systems and their applications in AAB, we carried out a comprehensive literature search using the Web of Science Core Collection database. The Acetobacteraceae family currently comprises 49 genera. We found overall 6097 publications related to one or more AAB genera since 1973, when the first successful recombinant DNA experiments in Escherichia coli have been published. The use of plasmids in AAB began in 1985 and till today was reported for only nine out of the 49 AAB genera currently described. We found at least five major expression plasmid lineages and a multitude of further expression plasmids, almost all enabling only constitutive target gene expression. Only recently, two regulatable expression systems became available for AAB, an N-acyl homoserine lactone (AHL)-inducible system for Komagataeibacter rhaeticus and an l-arabinose-inducible system for Gluconobacter oxydans. Thus, after 35 years of constitutive target gene expression in AAB, we now have the first regulatable expression systems for AAB in hand and further regulatable expression systems for AAB can be expected. Key points • Literature search revealed developments and usage of expression systems in AAB. • Only recently 2 regulatable plasmid systems became available for only 2 AAB genera. • Further regulatable expression systems for AAB are in sight.

scholarly journals Microrna-130a Downregulates HCV Replication through an atg5-Dependent Autophagy Pathway

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 338 ◽  
Author(s):  
Xiaoqiong Duan ◽  
Xiao Liu ◽  
Wenting Li ◽  
Jacinta A. Holmes ◽  
Annie J. Kruger ◽  
...  
Keyword(s):  
Immune Responses ◽  
Binding Sites ◽  
Target Gene ◽  
Target Genes ◽  
Pyruvate Metabolism ◽  
Host Immune Responses ◽  
Qrt Pcr ◽  
Antiviral Genes ◽  
Autophagy Pathway ◽  
Host Antiviral Response

We previously identified that miR-130a downregulates HCV replication through two independent pathways: restoration of host immune responses and regulation of pyruvate metabolism. In this study, we further sought to explore host antiviral target genes regulated by miR-130a. We performed a RT² Profiler™ PCR array to identify the host antiviral genes regulated by miR-130a. The putative binding sites between miR-130a and its downregulated genes were predicted by miRanda. miR-130a and predicted target genes were over-expressed or knocked down by siRNA or CRISPR/Cas9 gRNA. Selected gene mRNAs and their proteins, together with HCV replication in JFH1 HCV-infected Huh7.5.1 cells were monitored by qRT-PCR and Western blot. We identified 32 genes that were significantly differentially expressed more than 1.5-fold following miR-130a overexpression, 28 of which were upregulated and 4 downregulated. We found that ATG5, a target gene for miR-130a, significantly upregulated HCV replication and downregulated interferon stimulated gene expression. miR-130a downregulated ATG5 expression and its conjugation complex with ATG12. ATG5 and ATG5-ATG12 complex affected interferon stimulated gene (ISG) such as MX1 and OAS3 expression and subsequently HCV replication. We concluded that miR-130a regulates host antiviral response and HCV replication through targeting ATG5 via the ATG5-dependent autophagy pathway.

scholarly journals LPS-induced expression of CD14 in the TRIF pathway is epigenetically regulated by sulforaphane in porcine pulmonary alveolar macrophages

2016 ◽  
Vol 22 (8) ◽  
pp. 682-695 ◽  
Author(s):  
Qin Yang ◽  
Maren J Pröll ◽  
Dessie Salilew-Wondim ◽  
Rui Zhang ◽  
Dawit Tesfaye ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alveolar Macrophages ◽  
Methylation Status ◽  
Gene Body ◽  
Gene Body Methylation ◽  
Tnf Α ◽  
Pulmonary Alveolar Macrophages ◽  
Cluster Of Differentiation ◽  
Cd14 Gene

Pulmonary alveolar macrophages (AMs) are important in defense against bacterial lung inflammation. Cluster of differentiation 14 (CD14) is involved in recognizing bacterial lipopolysaccharide (LPS) through MyD88-dependent and TRIF pathways of innate immunity. Sulforaphane (SFN) shows anti-inflammatory activity and suppresses DNA methylation. To identify CD14 epigenetic changes by SFN in the LPS-induced TRIF pathway, an AMs model was investigated in vitro. CD14 gene expression was induced by 5 µg/ml LPS at the time point of 12 h and suppressed by 5 µM SFN. After 12 h of LPS stimulation, gene expression was significantly up-regulated, including TRIF, TRAF6, NF-κB, TRAF3, IRF7, TNF-α, IL-1β, IL-6, and IFN-β. LPS-induced TRAM, TRIF, RIPK1, TRAF3, TNF-α, IL-1β and IFN-β were suppressed by 5 µM SFN. Similarly, DNMT3a expression was increased by LPS but significantly down-regulated by 5 µM SFN. It showed positive correlation of CD14 gene body methylation with in LPS-stimulated AMs, and this methylation status was inhibited by SFN. This study suggests that SFN suppresses CD14 activation in bacterial inflammation through epigenetic regulation of CD14 gene body methylation associated with DNMT3a. The results provide insights into SFN-mediated epigenetic down-regulation of CD14 in LPS-induced TRIF pathway inflammation and may lead to new methods for controlling LPS-induced inflammation in pigs.

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