scholarly journals A chemical method to sequence 5-formylcytosine on RNA

2021 ◽  
Author(s):  
Ang Li ◽  
Xuemeng Sun ◽  
A Emilia Arguello ◽  
Ralph E Kleiner
Keyword(s):  
Chemical Method ◽  
Pcr Amplification ◽  
Biological Processes ◽  
Rna Modifications ◽  
Eukaryotic Evolution ◽  
Mitochondrial Trna ◽  
Wobble Position ◽  
Nucleotide Resolution ◽  
High Level ◽  
C To T Mutations

Epitranscriptomic RNA modifications can regulate biological processes, but there remains a major gap in our ability to identify and measure individual modifications at nucleotide resolution. Here we present Mal-Seq, a chemical method to sequence 5-formylcytosine (f5C) modifications on RNA based upon selective and efficient malononitrile-mediated labeling of f5C residues to generate adducts that are read as C-to-T mutations upon reverse transcription and PCR amplification. We apply Mal-Seq to characterize the prevalence of f5C at the wobble position of mt-tRNA(Met) in different organisms and tissue types and find that high-level f5C modification is present in mammals but lacking in lower eukaryotes. Our work sheds light on mitochondrial tRNA modifications throughout eukaryotic evolution and provides a general platform for characterizing the f5C epitranscriptome.

scholarly journals Transcriptome Analysis Provides Insights into the Mechanism of Astaxanthin Enrichment in a Mutant of the Ridgetail White Prawn Exopalaemon carinicauda

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 618
Author(s):  
Yue Jin ◽  
Shihao Li ◽  
Yang Yu ◽  
Chengsong Zhang ◽  
Xiaojun Zhang ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Underlying Mechanism ◽  
Biological Processes ◽  
Wild Type ◽  
Expression Levels ◽  
In The Wild ◽  
Cuticle Proteins ◽  
Apolipoprotein D ◽  
High Level ◽  
Lower Expression

A mutant of the ridgetail white prawn, which exhibited rare orange-red body color with a higher level of free astaxanthin (ASTX) concentration than that in the wild-type prawn, was obtained in our lab. In order to understand the underlying mechanism for the existence of a high level of free astaxanthin, transcriptome analysis was performed to identify the differentially expressed genes (DEGs) between the mutant and wild-type prawns. A total of 78,224 unigenes were obtained, and 1863 were identified as DEGs, in which 902 unigenes showed higher expression levels, while 961 unigenes presented lower expression levels in the mutant in comparison with the wild-type prawns. Based on Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis, as well as further investigation of annotated DEGs, we found that the biological processes related to astaxanthin binding, transport, and metabolism presented significant differences between the mutant and the wild-type prawns. Some genes related to these processes, including crustacyanin, apolipoprotein D (ApoD), cathepsin, and cuticle proteins, were identified as DEGs between the two types of prawns. These data may provide important information for us to understand the molecular mechanism of the existence of a high level of free astaxanthin in the prawn.

scholarly journals Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing

2019 ◽  
Vol 47 (20) ◽  
pp. e126-e126 ◽  
Author(s):  
Christel Enroth ◽  
Line Dahl Poulsen ◽  
Søren Iversen ◽  
Finn Kirpekar ◽  
Anders Albrechtsen ◽  
...  
Keyword(s):  
Statistical Significance ◽  
Yeast Cells ◽  
Alignment Error ◽  
Rna Modifications ◽  
Abasic Sites ◽  
Mutational Profiling ◽  
Domains Of Life ◽  
High Throughput Detection ◽  
Nucleotide Resolution ◽  
And Control

Abstract Methylation of guanosine on position N7 (m7G) on internal RNA positions has been found in all domains of life and have been implicated in human disease. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications at nucleotide resolution. In our method, m7G modified positions are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription and sequenced. We detect positions with increased mutation rates in the reduced and control samples taking the possibility of sequencing/alignment error into account and use replicates to calculate statistical significance based on log likelihood ratio tests. We show that m7G-MaP-seq efficiently detects known m7G modifications in rRNA with mutational rates up to 25% and we map a previously uncharacterised evolutionarily conserved rRNA modification at position 1581 in Arabidopsis thaliana SSU rRNA. Furthermore, we identify m7G modifications in budding yeast, human and arabidopsis tRNAs and demonstrate that m7G modification occurs before tRNA splicing. We do not find any evidence for internal m7G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA, including human Let-7e. Likewise, high sequencing depth m7G-MaP-seq analysis of mRNA from E. coli or yeast cells did not identify any internal m7G modifications.

scholarly journals Nucleotide resolution profiling of m3C RNA modification by HAC-seq

2020 ◽  
Author(s):  
Jia Cui ◽  
Qi Liu ◽  
Erdem Sendinc ◽  
Yang Shi ◽  
Richard I Gregory
Keyword(s):  
Rna Modification ◽  
Rna Modifications ◽  
Mcf7 Cells ◽  
Single Nucleotide ◽  
Novel Method ◽  
Specific Mapping ◽  
Nucleotide Resolution ◽  
And Function ◽  
Trna Isoacceptors ◽  
Single Nucleotide Resolution

Abstract Cellular RNAs are subject to a myriad of different chemical modifications that play important roles in controlling RNA expression and function. Dysregulation of certain RNA modifications, the so-called ‘epitranscriptome’, contributes to human disease. One limitation in studying the functional, physiological, and pathological roles of the epitranscriptome is the availability of methods for the precise mapping of individual RNA modifications throughout the transcriptome. 3-Methylcytidine (m3C) modification of certain tRNAs is well established and was also recently detected in mRNA. However, methods for the specific mapping of m3C throughout the transcriptome are lacking. Here, we developed a m3C-specific technique, Hydrazine-Aniline Cleavage sequencing (HAC-seq), to profile the m3C methylome at single-nucleotide resolution. We applied HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs in human cells. We found that tRNAs are the predominant m3C-modified RNA species, with 17 m3C modification sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors in MCF7 cells. We found no evidence for m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs in these cells. HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m3C RNA modification at single-nucleotide resolution, and could be widely applied to reveal the m3C methylome in different cells and tissues.

scholarly journals Genetic Diversity of Some Sweet Cherry Cultivars Based on Molecular Markers

2016 ◽  
Vol 73 (2) ◽  
pp. 153
Author(s):  
Ioana Virginia Berindean ◽  
Elena Tămaş ◽  
Oana Maria Toderic ◽  
Ioan Zagrai
Keyword(s):  
Tree Species ◽  
Rapd Markers ◽  
Sweet Cherry ◽  
Prunus Avium ◽  
Molecular Level ◽  
Genetic Relationships ◽  
Pcr Amplification ◽  
High Level ◽  
Sweet Cherry Cultivars

Sweet cherry (Prunus avium L.), originated around the Caspian and Black Sea, is an important fruit tree species of economic interest, and hence, breeding and conservation are requested (. Genetic analysis at the molecular level can be used effectively to study molecular polymorphism existing between intraspecific and interspecific tree species and phylogenetic relationships between them and their hybrids. The purpose of this study was to characterize and determine genetic relationships among the sweet cherry native genotypes belonging to Fruit Research & Development Station Bistrita, Romania, using RAPD markers. To eliminate the existence of possible synonyms from national romanian collection, we collect four Van cultivars, from four different national collection. For molecular analysis of the 16 varieties of sweet cherry were considered 13 RAPD primers selected from the literature. They were later used to determine the genetic variability at the molecular level using PAST program, and the dendrogram was generated based on Jaccard’s genetic distance. The dendrogram constructed by PAST software. The quantity and quality of the DNA obtained was suitable to achieve PCR amplification step. Only seven out of the 13 RAPD primers have generate polymorphic bands. The rest of seven were monomorphics. The most polymorphic primer was OPB10 which generated 11 bands from which 100% were polymorphic.Seven RAPD primers generated a high level of polymorphism which allowed to divide these cherry varieties into two groups according to their genetic geographical origin and the pedigree.

scholarly journals Genetic Characterization of Highly Fluoroquinolone-Resistant Clinical Escherichia coli Strains from China: Role ofacrR Mutations

2001 ◽  
Vol 45 (5) ◽  
pp. 1515-1521 ◽  
Author(s):  
Hui Wang ◽  
Joann L. Dzink-Fox ◽  
Minjun Chen ◽  
Stuart B. Levy
Keyword(s):  
Escherichia Coli ◽  
Blot Analysis ◽  
Genetic Basis ◽  
Efflux Pump ◽  
Pcr Amplification ◽  
Fluoroquinolone Resistance ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
High Level

ABSTRACT The genetic basis for fluoroquinolone resistance was examined in 30 high-level fluoroquinolone-resistant Escherichia coliclinical isolates from Beijing, China. Each strain also demonstrated resistance to a variety of other antibiotics. PCR sequence analysis of the quinolone resistance-determining region of the topoisomerase genes (gyrA/B, parC) revealed three to five mutations known to be associated with fluoroquinolone resistance. Western blot analysis failed to demonstrate overexpression of MarA, and Northern blot analysis did not detect overexpression of soxS RNA in any of the clinical strains. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in 19 of 30 strains of E. colitested, and all 19 strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of eight isolates revealed amino acid changes in four isolates, a 9-bp deletion in another, and a 22-bp duplication in a sixth strain. Complementation with a plasmid-borne wild-type acrR gene reduced the level of AcrA in the mutants and partially restored antibiotic susceptibility 1.5- to 6-fold. This study shows that mutations in acrR are an additional genetic basis for fluoroquinolone resistance.

scholarly journals Novel Tetracycline Resistance Gene,tet(32), in the Clostridium-Related Human Colonic Anaerobe K10 and Its Transmission In Vitro to the Rumen Anaerobe Butyrivibrio fibrisolvens

2001 ◽  
Vol 45 (11) ◽  
pp. 3246-3249 ◽  
Author(s):  
Claire M. Melville ◽  
Karen P. Scott ◽  
Derry K. Mercer ◽  
Harry J. Flint
Keyword(s):  
Resistance Gene ◽  
Pcr Amplification ◽  
Tetracycline Resistance ◽  
Amino Acid Identity ◽  
Tetracycline Resistance Gene ◽  
Butyrivibrio Fibrisolvens ◽  
Acid Identity ◽  
High Level ◽  
Porcine Feces

ABSTRACT A novel tetracycline resistance gene, designatedtet(32), which confers a high level of tetracycline resistance, was identified in the Clostridium-related human colonic anaerobe K10, which also carries tet(W).tet(32) was transmissible in vitro to the rumen anaerobeButyrivibrio fibrisolvens2221R. The predicted gene product oftet(32) has 76% amino acid identity with Tet(O). PCR amplification indicated that tet(32) is widely distributed in the ovine rumen and in porcine feces.

Study on Automobile Painting-Wastewater Treatment with the Process Technology of Pretreatment-Air Flotation-SBR

2012 ◽  
Vol 550-553 ◽  
pp. 2237-2240 ◽  
Author(s):  
Yu Ping Tian ◽  
Shuang Shuang Kong
Keyword(s):  
Wastewater Treatment ◽  
Chemical Method ◽  
Removal Rate ◽  
Biological Method ◽  
Wastewater Discharge ◽  
Process Technology ◽  
Chemical Agents ◽  
Air Flotation ◽  
Primary Standards ◽  
High Level

Wastewater from automobile painting is characterized by complicated composition, high level of CODCr and poor biodegradability. In this experiment, it combines the physic-chemical method with the biological method together. At first, it pretreats the degreased wastewater and painting wastewater with chemical agents, after which the removal rate of the degreased wastewater is more than 50% and that of the painting wastewater is 60~80%. Then, the effluents together with electrophoresis wastewater were treated by the coagulation-flotation and SBR process technology, after which the removal rate of CODCr can reach over 80%, so as to achieve the primary standards (CODCr≤100mg/L; pH=7.0~7.5) of "Integrated Wastewater Discharge Standard" (GB8978-1996).

Genetic diversity and relationships among Secale L. based on RAMP markers

2004 ◽  
Vol 1 (2) ◽  
pp. 73-78 ◽  
Author(s):  
Shang Hai-Ying ◽  
Zheng You-Liang ◽  
Wei Yu-Ming ◽  
Wu Wei ◽  
Yan Ze-Hong
Keyword(s):  
Genetic Diversity ◽  
Genetic Relationships ◽  
Pcr Amplification ◽  
Group Method ◽  
Transitional Region ◽  
Arithmetic Average ◽  
Pair Group ◽  
Broad Leaf ◽  
Polymorphic Bands ◽  
High Level

AbstractGenetic diversity and relationships among 21 accessions of Secale L., including three species and 10 subspecies, were evaluated using RAMP markers. Forty-one out of 80 (50.5%) RAMP primers, which produced clear and polymorphic bands, were selected for PCR amplification of genomic DNA. A total of 446 bands were amplified from the 41 primers, and 428 of these bands (about 96%) were polymorphic. Three to 19 polymorphic bands could be amplified from each primer, with an average of 10.4 bands. The RAMP-based genetic similarity (GS) values among the 21 Secale accessions ranged from 0.266 to 0.658, with a mean of 0.449. A high level of genetic variation was found between or within the wild populations and the cultivars. Based on the GS matrix, a dendrogram was constructed using the unweighted pair group method with arithmetic average (UPGMA). All 21 accessions could be distinguished by RAMP markers. Clustering results showed that the genetic diversity of Secale based on RAMP markers was correlated with geographical distribution. Six rye cultivars, originating from Poland, Portugal, Mexico, Hungary, Armenia and Ukraine, were clustered into one group. The six countries are all located in the transitional region of broad-leaf forests between maritime and continental temperate zones, with narrow latitude span. In comparison, the other five cultivars from countries scattered over a region with large latitude span were distributed within different groups or subgroups. Genetic relationships based on RAMP markers had great deviation from the original taxonomy. Some subspecies of the same species were distributed within different groups, while some accessions of different species were closely clustered into one subgroup. These results suggest that RAMP markers could be an effective technique for detecting genetic diversity among Secale and give some useful information about its phylogenic relationships.

scholarly journals A single editing event is a prerequisite for efficient processing of potato mitochondrial phenylalanine tRNA.

1996 ◽  
Vol 16 (7) ◽  
pp. 3504-3510 ◽  
Author(s):  
L Marechal-Drouard ◽  
A Cosset ◽  
C Remacle ◽  
D Ramamonjisoa ◽  
A Dietrich
Keyword(s):  
Plant Mitochondria ◽  
Pcr Amplification ◽  
Mitochondrial Trna ◽  
Processing Efficiency ◽  
Editing Event ◽  
Tertiary Structures ◽  
Reverse Transcription Pcr ◽  
Efficient Processing ◽  
Trna Editing

In bean, potato, and Oenothera plants, the C encoded at position 4 (C4) in the mitochondrial tRNA Phe GAA gene is converted into a U in the mature tRNA. This nucleotide change corrects a mismatched C4-A69 base pair which appears when the gene sequence is folded into the cloverleaf structure. C-to-U conversions constitute the most common editing events occurring in plant mitochondrial mRNAs. While most of these conversions introduce changes in the amino acids specified by the mRNA and appear to be essential for the synthesis of functional proteins in plant mitochondria, the putative role of mitochondrial tRNA editing has not yet been defined. Since the edited form of the tRNA has the correct secondary and tertiary structures compared with the nonedited form, the two main processes which might be affected by a nucleotide conversion are aminoacylation and maturation. To test these possibilities, we determined the aminoacylation properties of unedited and edited potato mitochondrial tRNAPhe in vitro transcripts, as well as the processing efficiency of in vitro-synthesized potato mitochondrial tRNAPhe precursors. Reverse transcription-PCR amplification of natural precursors followed by cDNA sequencing was also used to investigate the influence of editing on processing. Our results show that C-to-U conversion at position 4 in the potato mitochondrial tRNA Phe GAA is not required for aminoacylation with phenylalanine but is likely to he essential for efficient processing of this tRNA.

scholarly journals Development and Application of Genic Simple Sequence Repeat Markers from the Transcriptome of Loquat

2014 ◽  
Vol 139 (5) ◽  
pp. 507-517 ◽  
Author(s):  
Xiaoying Li ◽  
Hongxia Xu ◽  
Jianjun Feng ◽  
Junwei Chen
Keyword(s):  
Large Scale ◽  
Trinucleotide Repeat ◽  
Genetic Relationships ◽  
Pcr Amplification ◽  
Eriobotrya Japonica ◽  
Pcr Products ◽  
Ssr Loci ◽  
Trait Locus ◽  
High Level ◽  
Simple Sequence

Deep transcriptome sequencing allows for the acquisition of large-scale microsatellite information, and it is especially useful for genetic diversity analysis and mapping in plants without reference genome sequences. In this study, a total of 14,004 simple sequence repeats (SSRs) were mined from 10,511 unigenes screening of 63,608 nonredundant transcriptome unigenes in loquat (Eriobotrya japonica) with a frequency of 22 SSR loci distributed over 100 unigenes. Dinucleotide and trinucleotide repeat SSRs were dominant, accounting for 20.62%, and 42.1% of the total, respectively. Seventy primer pairs were designed from partial SSRs and used for polymerase chain reaction (PCR) amplification. Of these primer pairs, 54 exhibited amplification and 33 were polymorphic. The number of alleles at these loci ranged from two to 17, and the polymorphism information content values ranged from 0.24 to 0.89. We tested the transferability of 33 SSR polymorphic primer pairs in apple and pear, and the transferability rates in these two species were 90.9% and 87.9%, respectively. A high level of marker polymorphism was observed in apple [Malus ×domestica (66.7%)], whereas a low level was observed in pear [Pyrus sp. (51.5%)]. In addition, the PCR products from seven SSR primer pairs were selected for sequence analysis, and 89.2% of the fragments were found to contain SSRs. SSR motifs were conserved among loquat, apple, and pear. According to our sequencing results for real SSR loci, ≈12,490 SSR loci were present in these loquat unigenes. The cluster dendrogram showed a distinct separation into different groups for these three species, indicating that these SSR markers were useful in the evaluation of genetic relationships and diversity between and within the species of Maloideae in the Rosaceae. The results of our identified SSRs should be useful for genetic linkage map construction, quantitative trait locus mapping, and molecular marker-assisted breeding of loquat and related species.

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