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1.
Proc Natl Acad Sci U S A ; 119(19): e2203967119, 2022 May 10.
Article in English | MEDLINE | ID: mdl-35503911

ABSTRACT

SignificanceH-DNA is a non-B-form DNA structure containing intramolecular triplex and single-stranded DNA (ssDNA) regions. H-DNA-forming motifs include polypyrimidine•polypurine mirror repeat sequences, which occur frequently in eukaryotic genomes. These motifs have biological impacts on genome stability and processes such as replication and transcription, but their non-B DNA-forming potentials are not fully understood. Here, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated by a deep-sequencing technology that uses the ssDNA-specific nuclease S1 to detect ssDNA. It is currently unclear whether the H-DNA detected formed in vitro or was already present in vivo. Nevertheless, this study provides an approach to unveiling structural features of intramolecular triplexes genome-wide at high spatial resolution.


Subject(s)
Genome , Nucleotide Motifs , Animals , Base Sequence , Genome/genetics , Mice , Nucleic Acid Conformation
3.
Nat Commun ; 13(1): 2424, 2022 May 03.
Article in English | MEDLINE | ID: mdl-35505047

ABSTRACT

Mass spectrometry is an important method for analysis of modified nucleosides ubiquitously present in cellular RNAs, in particular for ribosomal and transfer RNAs that play crucial roles in mRNA translation and decoding. Furthermore, modifications have effect on the lifetimes of nucleic acids in plasma and cells and are consequently incorporated into RNA therapeutics. To provide an analytical tool for sequence characterization of modified RNAs, we developed Pytheas, an open-source software package for automated analysis of tandem MS data for RNA. The main features of Pytheas are flexible handling of isotope labeling and RNA modifications, with false discovery rate statistical validation based on sequence decoys. We demonstrate bottom-up mass spectrometry characterization of diverse RNA sequences, with broad applications in the biology of stable RNAs, and quality control of RNA therapeutics and mRNA vaccines.


Subject(s)
RNA , Tandem Mass Spectrometry , Base Sequence , RNA/chemistry , RNA, Transfer/chemistry , Software , Tandem Mass Spectrometry/methods
4.
BMC Bioinformatics ; 23(1): 155, 2022 May 02.
Article in English | MEDLINE | ID: mdl-35501677

ABSTRACT

BACKGROUND: Recent deep sequencing technologies have proven to be valuable resources to gain insights into the expression profiles of diverse tRNAs. However, despite these technologies, the association of tRNAs with diverse diseases has not been explored in depth because analytical tools are lacking. RESULTS: We developed a user-friendly tool, tRNA Expression Analysis Software Utilizing R for Easy use (tReasure), to analyze differentially expressed tRNAs (DEtRNAs) from deep sequencing data of small RNAs using R packages. tReasure can quantify individual mature tRNAs, isodecoders, and isoacceptors. By adopting stringent mapping strategies, tReasure supports the precise measurement of mature tRNA read counts. The whole analysis workflow for determining DEtRNAs (uploading FASTQ files, removing adapter sequences and poor-quality reads, mapping and quantifying tRNAs, filtering out low count tRNAs, determining DEtRNAs, and visualizing statistical analysis) can be performed with the tReasure package. CONCLUSIONS: tReasure is an open-source software available for download at https://treasure.pmrc.re.kr and will be indispensable for users who have little experience with command-line software to explore the biological implication of tRNA expression.


Subject(s)
RNA , Software , Base Sequence , RNA, Transfer/genetics , Sequence Analysis, RNA
5.
Exp Suppl ; 114: 43-69, 2022.
Article in English | MEDLINE | ID: mdl-35543998

ABSTRACT

The microsporidia are a phylum of intracellular parasites that represent the eukaryotic cell in a state of extreme reduction, with genomes and metabolic capabilities embodying eukaryotic cells in arguably their most streamlined state. Over the past 20 years, microsporidian genomics has become a rapidly expanding field starting with sequencing of the genome of Encephalitozoon cuniculi, one of the first ever sequenced eukaryotes, to the current situation where we have access to the data from over 30 genomes across 20+ genera. Reaching back further in evolutionary history, to the point where microsporidia diverged from other eukaryotic lineages, we now also have genomic data for some of the closest known relatives of the microsporidia such as Rozella allomycis, Metchnikovella spp. and Amphiamblys sp. Data for these organisms allow us to better understand the genomic processes that shaped the emergence of the microsporidia as a group. These intensive genomic efforts have revealed some of the processes that have shaped microsporidian cells and genomes including patterns of genome expansions and contractions through gene gain and loss, whole genome duplication, differential patterns of invasion and purging of transposable elements. All these processes have been shown to occur across short and longer time scales to give rise to a phylum of parasites with dynamic genomes with a diversity of sizes and organisations.


Subject(s)
Microsporidia , Base Sequence , Evolution, Molecular , Genome, Fungal/genetics , Genomics , Microsporidia/genetics
6.
Methods Mol Biol ; 2477: 331-348, 2022.
Article in English | MEDLINE | ID: mdl-35524126

ABSTRACT

Base editing is a CRISPR-Cas9 genome engineering tool that allows programmable mutagenesis without the creation of double-stranded breaks. Here, we describe the design and execution of large-scale base editing screens using the Target-AID base editor in yeast. Using this approach, thousands of sites can be mutated simultaneously. The effects of these mutations on fitness can be measured using a pooled growth competition assay followed by DNA sequencing of gRNAs as barcodes.


Subject(s)
CRISPR-Cas Systems , Gene Editing , Base Sequence , CRISPR-Cas Systems/genetics , Mutagenesis/genetics , RNA, Guide/genetics
7.
Trop Anim Health Prod ; 54(3): 183, 2022 May 07.
Article in English | MEDLINE | ID: mdl-35525911

ABSTRACT

Feather colours are used by avian species for defense, adaptation and signaling. Melanocortin-1 receptor (MC1R) gene is one of the genes responsible for feather colour. This study identified selection signatures in MC1R gene of Nigerian indigenous turkeys (NIT) using British United turkeys (BUT) as control breed to investigate the evolutionary processes that have shaped NIT with various feather colours. Complete MC1R gene of 146 NIT (76 males and 70 females) and 32 BUT (18 males and 14 females) were sequenced. Transition/transversion and codon usage biases were predicted using MEGA v6 software. The selective force acting on the gene was predicted using HyPhy software. The FST values were estimated using Arlequin v3.5. The highest transition/transversion bias was predicted for white BUT (1.00) while the lowest was predicted for black NIT (0.50). Negative dN-dS values, indicative of purifying selection, were observed in MC1R gene of all the turkeys. The highest pairwise FST was observed between the MC1R gene of white BUT and black NIT while the least was observed between lavender NIT and white NIT. No recombination event was observed in black NIT and white BUT. The relative synonymous codon usage was the same among different colours for some codons. Presence of purifying selection in MC1R gene of all the turkeys with different feather colours confirms that the gene plays role in many biological processes such as feather colouration, behaviour, pain perception, immunity, growth and adaptation. The results also suggested that the genetic mechanisms generating different feather colours in turkeys are conserved.


Subject(s)
Receptor, Melanocortin, Type 1 , Turkeys , Animals , Base Sequence , Feathers , Female , Male , Plant Breeding , Receptor, Melanocortin, Type 1/genetics , Turkeys/genetics
8.
Sci Rep ; 12(1): 5944, 2022 Apr 08.
Article in English | MEDLINE | ID: mdl-35396527

ABSTRACT

Heterogeneous Ribonucleoprotein D (hnRNPD) is an RNA binding protein involved in post-transcriptional regulation of multiple mediators of carcinogenesis. We previously demonstrated a strong association of hnRNPD over expression with poor outcome in Oral Squamous Cell Carcinoma (OSCC). However, hitherto the precise molecular mechanism of its overexpression in oral cancer was not clear. Therefore, in an attempt to elucidate the transcriptional regulation of hnRNPD expression, we cloned 1406 bp of 5' flanking region of human hnRNPD gene along with 257 bp of its first exon upstream to promoterless luciferase reporter gene in pGL3-Basic. Transfection of the resulting construct in SCC-4 cells yielded 1271 fold higher luciferase activity over parent vector. By promoter deletion analysis, we identified a canonical TATA box containing 126 bp core promoter region that retained ~ 58% activity of the full length promoter. In silico analysis revealed the presence of four putative NFκB binding motifs in the promoter. Sequential deletion of these motifs from the full-length promoter reporter construct coupled with luciferase assays revealed an 82% decrease in promoter activity after deletion of the first (-1358/-1347) motif and 99% reduction after the deletion of second motif (-1052/-1041). In-vivo binding of NFκB (RelA) to these two motifs in SCC-4 cells was confirmed by ChIP assays. Site directed mutagenesis of even one of these two motifs completely abolished promoter activity, while mutagenesis of the remaining two motifs had marginal effect on the same. Consistent with these findings, treatment of SCC-4 cells with PDTC, a known inhibitor of NFκB dramatically reduced the levels hnRNPD mRNA and protein. Finally, the expression of hnRNPD and NFκB in clinical specimen from 37 oral cancer patients was assessed and subjected to Spearmen's Correlation analysis which revealed a strong positive correlation between the two. Thus, results of the present study for the first time convincingly demonstrate NFκB (RelA) mediated transcriptional upregulation of hnRNPD expression in oral cancer.


Subject(s)
Carcinoma, Squamous Cell , Heterogeneous Nuclear Ribonucleoprotein D0 , Mouth Neoplasms , Transcription Factor RelA , Base Sequence , Carcinoma, Squamous Cell/genetics , Heterogeneous Nuclear Ribonucleoprotein D0/genetics , Humans , Luciferases , Mouth Neoplasms/genetics , NF-kappa B/genetics , NF-kappa B/metabolism , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Transcriptional Activation
9.
ACS Sens ; 7(4): 1132-1137, 2022 Apr 22.
Article in English | MEDLINE | ID: mdl-35412319

ABSTRACT

We describe an electrochemical strategy to transduce allosteric transcription factor (aTF) binding affinity to sense steroid hormones. Our approach utilizes square wave voltammetry to monitor changes in current output as a progesterone (PRG)-specific aTF (SRTF1) unbinds from the cognate DNA sequence in the presence of PRG. The sensor detects PRG in artificial urine samples with sufficient sensitivity suitable for clinical applications. Our results highlight the capability of using aTFs as the biorecognition elements to develop electrochemical point-of-care biosensors for the detection of small-molecule biomarkers and analytes.


Subject(s)
Biosensing Techniques , Progesterone , Base Sequence , Biosensing Techniques/methods , DNA/genetics , Transcription Factors/genetics , Transcription Factors/metabolism
10.
BMC Ophthalmol ; 22(1): 172, 2022 Apr 15.
Article in English | MEDLINE | ID: mdl-35428228

ABSTRACT

BACKGROUND: To identify the genetic mutation of a four-generation autosomal dominant congenital cataract family in China. METHODS: Targeted region sequencing containing 778 genes associated with ocular diseases was performed to screen for the potential mutation, and Sanger sequencing was used to confirm the mutation. The homology model was constructed to identify the protein structural change, several online software were used to predict the mutation impact. CLUSTALW was used to perform multiple sequence alignment from different species. RESULTS: A novel heterozygous mutation, GJA8 NM_005267.5: c.124G > A, p.(E42K) was found, which cosegregated with congenital cataract phenotype in this family. Bioinformatics analysis of the mutation showed that the surface potential diagram of proteins changed. Several online programs predicted the mutation was 'Pathogenic', 'Damaging', 'Disease causing' or 'Deleterious'. CONCLUSIONS: A novel mutation NM_005267.5(GJA8):c.124G > A was identified in our study. Our finding can broaden the mutation spectrum of GJA8, enrich the phenotype-genotype correlation of congenital cataract and help to better understand the genetic background of congenital cataract.


Subject(s)
Cataract , Connexins , Base Sequence , Cataract/congenital , Cataract/genetics , Connexins/genetics , DNA Mutational Analysis , Humans , Mutation , Mutation, Missense , Pedigree
11.
Genome Biol ; 23(1): 103, 2022 Apr 21.
Article in English | MEDLINE | ID: mdl-35449021

ABSTRACT

Recent progress in deep learning has greatly improved the prediction of RNA splicing from DNA sequence. Here, we present Pangolin, a deep learning model to predict splice site strength in multiple tissues. Pangolin outperforms state-of-the-art methods for predicting RNA splicing on a variety of prediction tasks. Pangolin improves prediction of the impact of genetic variants on RNA splicing, including common, rare, and lineage-specific genetic variation. In addition, Pangolin identifies loss-of-function mutations with high accuracy and recall, particularly for mutations that are not missense or nonsense, demonstrating remarkable potential for identifying pathogenic variants.


Subject(s)
Pangolins , RNA Splicing , Animals , Base Sequence , Mutation , RNA Splice Sites
12.
Methods Mol Biol ; 2484: 291-311, 2022.
Article in English | MEDLINE | ID: mdl-35461459

ABSTRACT

RNA sequencing (RNA-seq) data is by now the most common method to study differential gene expression. Here we present a pipeline from RNA-seq generation to analysis with examples based on our own barley anther and meiocyte transcriptome. The bioinformatics pipeline will give everyone, from a beginner to a more experienced user, the possibility to analyze their datasets and identify significantly differentially expressed genes. It also allows differential alternative splicing analysis which will become increasingly common due to the high regulatory impact on the gene expression. We describe use of the Galaxy interface for RNA-seq read quantification and the 3D RNA-seq app for the downstream data analysis.


Subject(s)
Hordeum , Base Sequence , Data Analysis , Gene Expression Profiling/methods , High-Throughput Nucleotide Sequencing , Hordeum/genetics , Hordeum/metabolism , RNA/genetics , Sequence Analysis, RNA , Transcriptome
13.
J Chem Phys ; 156(16): 164907, 2022 Apr 28.
Article in English | MEDLINE | ID: mdl-35489993

ABSTRACT

Melting of DNA sequences may occur through a few major intermediate states, whose influence on the melting curve has been discussed previously, while their effect on the kinetics has not been explored thoroughly. Here, we chose a simple DNA sequence, forming a hairpin in its native (zipped) state, and study it using molecular dynamic (MD) simulations and a model integrating the Gaussian network model with bond-binding energies-the Gaussian binding energy (GBE) model. We find two major partial denaturation states, a bubble state and a partial unzipping state. We demonstrate the influence of these two states on the closing-opening base pair dynamics, as probed by a tagged bond auto-correlation function (ACF). We argue that the latter is measured by fluorescence correlation spectroscopy experiments, in which one base of the pair is linked to a fluorescent dye, while the complementary base is linked to a quencher, similar to the experiment reported by Altan-Bonnet et al. [Phys. Rev. Lett. 90, 138101 (2003)]. We find that tagging certain base pairs at temperatures around the melting temperature results in a multi-step relaxation of the ACF, while tagging other base pairs leads to an effectively single-step relaxation, albeit non-exponential. Only the latter type of relaxation has been observed experimentally, and we suggest which of the other base pairs should be tagged in order to observe multi-step relaxation. We demonstrate that this behavior can be observed with other sequences and argue that the GBE can reliably predict these dynamics for very long sequences, where MD simulations might be limited.


Subject(s)
DNA , Molecular Dynamics Simulation , Base Pairing , Base Sequence , DNA/chemistry , Kinetics
14.
Nat Commun ; 13(1): 2304, 2022 Apr 28.
Article in English | MEDLINE | ID: mdl-35484104

ABSTRACT

The self-assembly of DNA-coated colloids into highly-ordered structures offers great promise for advanced optical materials. However, control of disorder, defects, melting, and crystal growth is hindered by the lack of a microscopic understanding of DNA-mediated colloidal interactions. Here we use total internal reflection microscopy to measure in situ the interaction potential between DNA-coated colloids with nanometer resolution and the macroscopic melting behavior. The range and strength of the interaction are measured and linked to key material design parameters, including DNA sequence, polymer length, grafting density, and complementary fraction. We present a first-principles model that screens and combines existing theories into one coherent framework and quantitatively reproduces our experimental data without fitting parameters over a wide range of DNA ligand designs. Our theory identifies a subtle competition between DNA binding and steric repulsion and accurately predicts adhesion and melting at a molecular level. Combining experimental and theoretical results, our work provides a quantitative and predictive approach for guiding material design with DNA-nanotechnology and can be further extended to a diversity of colloidal and biological systems.


Subject(s)
Colloids , DNA , Base Sequence , Colloids/chemistry , Crystallization , DNA/chemistry , Nanotechnology
15.
Genome Med ; 14(1): 44, 2022 Apr 28.
Article in English | MEDLINE | ID: mdl-35484572

ABSTRACT

Structural variants (SVs) are implicated in the etiology of Mendelian diseases but have been systematically underascertained owing to sequencing technology limitations. Long-read sequencing enables comprehensive detection of SVs, but approaches for prioritization of candidate SVs are needed. Structural variant Annotation and analysis (SvAnna) assesses all classes of SVs and their intersection with transcripts and regulatory sequences, relating predicted effects on gene function with clinical phenotype data. SvAnna places 87% of deleterious SVs in the top ten ranks. The interpretable prioritizations offered by SvAnna will facilitate the widespread adoption of long-read sequencing in diagnostic genomics. SvAnna is available at https://github.com/TheJacksonLaboratory/SvAnn a .


Subject(s)
Genomics , Base Sequence , Chromosome Mapping , Humans , Sequence Analysis, DNA , Virulence
16.
BMC Genomics ; 23(Suppl 1): 301, 2022 Apr 13.
Article in English | MEDLINE | ID: mdl-35418074

ABSTRACT

BACKGROUND: Nucleosome positioning is the precise determination of the location of nucleosomes on DNA sequence. With the continuous advancement of biotechnology and computer technology, biological data is showing explosive growth. It is of practical significance to develop an efficient nucleosome positioning algorithm. Indeed, convolutional neural networks (CNN) can capture local features in DNA sequences, but ignore the order of bases. While the bidirectional recurrent neural network can make up for CNN's shortcomings in this regard and extract the long-term dependent features of DNA sequence. RESULTS: In this work, we use word vectors to represent DNA sequences and propose three new deep learning models for nucleosome positioning, and the integrative model NP_CBiR reaches a better prediction performance. The overall accuracies of NP_CBiR on H. sapiens, C. elegans, and D. melanogaster datasets are 86.18%, 89.39%, and 85.55% respectively. CONCLUSIONS: Benefited by different network structures, NP_CBiR can effectively extract local features and bases order features of DNA sequences, thus can be considered as a complementary tool for nucleosome positioning.


Subject(s)
Deep Learning , Nucleosomes , Animals , Base Sequence , Caenorhabditis elegans/genetics , Drosophila melanogaster/genetics , Nucleosomes/genetics , Plant Extracts
17.
Genome Res ; 32(4): 599-607, 2022 Apr.
Article in English | MEDLINE | ID: mdl-35361624

ABSTRACT

The complete, ungapped sequence of the short arms of human acrocentric chromosomes (SAACs) is still unknown almost 20 years after the near completion of the Human Genome Project. Yet these short arms of Chromosomes 13, 14, 15, 21, and 22 contain the ribosomal DNA (rDNA) genes, which are of paramount importance for human biology. The sequences of SAACs show an extensive variation in the copy number of the various repetitive elements, the full extent of which is currently unknown. In addition, the full spectrum of repeated sequences, their organization, and the low copy number functional elements are also unknown. The Telomere-to-Telomere (T2T) Project using mainly long-read sequence technology has recently completed the assembly of the genome from a hydatidiform mole, CHM13, and has thus established a baseline reference for further studies on the organization, variation, functional annotation, and impact in human disorders of all the previously unknown genomic segments, including the SAACs. The publication of the initial results of the T2T Project will update and improve the reference genome for a better understanding of the evolution and function of the human genome.


Subject(s)
Chromosomes, Human , Genome, Human , Base Sequence , Chromosomes, Human/genetics , DNA, Ribosomal/genetics , Female , Human Genome Project , Humans , Pregnancy
18.
Invest Ophthalmol Vis Sci ; 63(4): 17, 2022 04 01.
Article in English | MEDLINE | ID: mdl-35472218

ABSTRACT

Background: The progression and recurrence of pterygium mainly occur due to the abnormal proliferation and migration of stromal pterygium fibroblasts. This research explores the aberrant expression of small nucleolar RNA U3 (U3 snoRNA) in pterygium and elucidates the molecular mechanisms of U3 snoRNA in pterygium development. Methods: Primary human conjunctival fibroblasts (HCFs) and human pterygium fibroblasts (HPFs) were separated and cultured from fresh conjunctiva grafts and pterygium tissues. The PLKO.1 lentiviral system and CRISPR/Cas9 recombinant construct were, respectively, used to overexpress and silence U3 snoRNA in HPFs and HCFs for further specific phenotype analysis. RNA-seq and TMT-labeled quantitative protein mass spectrometry were utilized to evaluate the effect of U3 snoRNA on mRNA transcripts and protein synthesis. Results: Reduced U3 snoRNA in pterygium promotes HCF or HPF cells' proliferation, migration, and cell cycle but has no significant effect on apoptosis. U3 snoRNA modulates 18S rRNA synthesis through shearing precursor ribosomal RNA 47S rRNA at the 5' external transcribed spacer (5' ETS). Moreover, the altered U3 snoRNA causes mRNA and protein differential expression in HCF or HPF cells. Conclusions: The atypical U3 snoRNA regulates the translation of specific proteins to exert a suppressive function in pterygium through modulating the 18S rRNA synthesis. Here, we uncover a novel insight into U3 snoRNA biology in the development of pterygium.


Subject(s)
Pterygium , RNA, Small Nucleolar , Base Sequence , Conjunctiva/abnormalities , Conjunctiva/metabolism , Humans , Pterygium/genetics , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/genetics , RNA, Ribosomal, 18S/metabolism , RNA, Small Nucleolar/chemistry , RNA, Small Nucleolar/genetics , RNA, Small Nucleolar/metabolism
19.
J Vis Exp ; (182)2022 Apr 11.
Article in English | MEDLINE | ID: mdl-35467656

ABSTRACT

RNA is a biopolymer present in all domains of life, and its interactions with other molecules and/or reactive species, e.g., DNA, proteins, ions, drugs, and free radicals, are ubiquitous. As a result, RNA undergoes various reactions that include its cleavage, degradation, or modification, leading to biologically relevant species with distinct functions and implications. One example is the oxidation of guanine to 7,8-dihydro-8-oxoguanine (8-oxoG), which may occur in the presence of reactive oxygen species (ROS). Overall, procedures that characterize such products and transformations are largely valuable to the scientific community. To this end, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry is a widely used method. The present protocol describes how to characterize RNA fragments formed after enzymatic treatment. The chosen model uses a reaction between RNA and the exoribonuclease Xrn-1, where enzymatic digestion is halted at oxidized sites. Two 20-nucleotide long RNA sequences [5'-CAU GAA ACA A(8-oxoG)G CUA AAA GU] and [5'-CAU GAA ACA A(8-oxoG)(8-oxoG) CUA AAA GU] were obtained via solid-phase synthesis, quantified by UV-vis spectroscopy, and characterized via MALDI-TOF. The obtained strands were then (1) 5'-phosphorylated and characterized via MALDI-TOF; (2) treated with Xrn-1; (3) filtered and desalted; (4) analyzed via MALDI-TOF. This experimental setup led to the unequivocal identification of the fragments associated with the stalling of Xrn-1: [5'-H2PO4-(8-oxoG)G CUA AAA GU], [5'-H2PO4-(8-oxoG)(8-oxoG) CUA AAA GU], and [5'-H2PO4-(8-oxoG) CUA AAA GU]. The described experiments were carried out with 200 picomols of RNA (20 pmol used for MALDI analyses); however, lower amounts may result in detectable peaks with spectrometers using laser sources with more power than the one used in this work. Importantly, the described methodology can be generalized and potentially extended to product identification for other processes involving RNA and DNA, and may aid in the characterization/elucidation of other biochemical pathways.


Subject(s)
DNA , RNA , Base Sequence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
20.
Sheng Wu Gong Cheng Xue Bao ; 38(4): 1576-1588, 2022 Apr 25.
Article in Chinese | MEDLINE | ID: mdl-35470628

ABSTRACT

In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes LguⅠ and BbvCⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of LguⅠ and BbvCⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB-welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.


Subject(s)
Sphingomonas , Base Sequence , Cloning, Molecular , Fermentation , Plasmids/genetics , Sphingomonas/genetics , Sphingomonas/metabolism
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