Genetic diversity of the S-type small subunit ribosomal RNA gene of Plasmodium knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia

Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype.Methods:Thirty-nine blood samples infected with P.knowlesi were collected in Sabah,Malaysian Borneo and Peninsular Malaysia.The S-type SSU rRNA gene was amplified using polymerase chain reaction,cloned into a vector,and sequenced.The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10.This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11.Results:Overall,the phylogenetic analysis showed no evidence of a geographical cluster of P.knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences.The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect.Haplotype sharing of the S-type SSU rRNA gene was observed between the P.knowlesi isolates in Sabah,Malaysian Borneo,but not between Sabah,Malaysian Borneo and Peninsular Malaysia.Conclusions:This study suggests that the S-type SSU rRNA gene of P.knowlesi isolates in Sabah,Malaysian Borneo,and Peninsular Malaysia has fewer polymorphic sites,representing the conservation of the gene.These features make the S-type SSU rRNA gene suitable for comparative studies,such as determining the evolutionary relationships and common ancestry among P.knowlesi species.

Testing complete plastomes and nuclear ribosomal DNA sequences for species identification in a taxonomically difficult bamboo genus Fargesia

Fargesia,the largest genus within the temperate bamboo tribe Arundinarieae,has more than 90 species mainly distributed in the mountains of Southwest China.The Fargesia bamboos are important components of the subalpine forest ecosystems that provide food and habitat for many endangered animals,including the giant panda.However,species-level identification of Fargesia is difficult.Moreover,the rapid radiation and slow molecular evolutionary rate of Fargesia pose a significant challenge to using DNA barcoding with standard plant barcodes(rbcL,matK,and ITS) in bamboos.With progress in the sequencing technologies,complete plastid genomes(plastomes) and nuclear ribosomal DNA(nrDNA)sequences have been proposed as organelle barcodes for species identification;however,these have not been tested in bamboos.We collected 196 individuals representing 62 species of Fargesia to comprehensively evaluate the discriminatory power of plastomes and nrDNA sequences compared to standard barcodes.Our analysis indicates that complete plastomes have substantially higher discriminatory power(28.6%) than standard barcodes(5.7%),whereas nrDNA sequences show a moderate improvement(65.4%) compared to ITS(47.2%).We also found that nuclear markers performed better than plastid markers,and ITS alone had higher discriminatory power than complete plastomes.The study also demonstrated that plastomes and nrDNA sequences can contribute to intrageneric phylogenetic resolution in Fargesia.However,neither of these sequences were able to discriminate all the sampled species,and therefore,more nuclear markers need to be identified.

Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity

Ribosomes are abundant,large RNA-protein complexes that are the sites of all protein synthesis in cells.Defects in ribosomal proteins(RPs),including proteoforms arising from genetic variations,alternative splicing of RNA transcripts,post-translational modifications and alterations of protein expression level,have been linked to a diverse range of diseases,including cancer and aging.Comprehensive characterization of ribosomal proteoforms is challenging but important for the discovery of potential disease biomarkers or protein targets.In the present work,using E.coli 70S RPs as an example,we first developed a top-down proteomics approach on a Waters Synapt G2 Si mass spectrometry(MS)system,and then applied it to the HeLa 80S ribosome.The results were complemented by a bottom-up approach.In total,50 out of 55 RPs were identified using the top-down approach.Among these,more than 30 RPs were found to have their N-terminal methionine removed.Additional modifications such as methylation,acetylation,and hydroxylation were also observed,and the modification sites were identified by bottomup MS.In a HeLa 80S ribosomal sample,we identified 98 ribosomal proteoforms,among which multiple truncated 80S ribosomal proteoforms were observed,the type of information which is often overlooked by bottom-up experiments.Although their relevance to diseases is not yet known,the integration of topdown and bottom-up proteomics approaches paves the way for the discovery of proteoform-specific disease biomarkers or targets.

Effects of electroacupuncture on the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of rats with vascular dementia

This study investigated the mechanism underlying electroacupuncture therapy for vascular dementia through electroacupuncture at the acupoints of Baihui （DU20）, Dazhui （DU14）, and bilateral Shenshu （BL23） in a rat model of vascular dementia produced by bilateral middle cerebral artery occlusion. Morris water maze test showed that electroacupuncture improved the learning ability of vascular dementia rats. Western blot assay revealed that the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in vascular dementia rats was significantly increased after electroacupuncture, compared with the model group that was not treated with acupuncture. The average escape latency was also shortened after electroacupuncture, and escape strategies in the spatial probe test improved from edge and random searches, to linear and trending swim pathways. The experimental findings indicate that electroacupuncture improves learning and memory ability by up-regulating expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of vascular dementia rats.

Complete nuclear ribosomal DNA sequence amplification and molecular analyses of Bangia (Bangiales, Rhodophyta) from China

Filamentous Bangia,which are distributed extensively throughout the world,have simple and similar morphological characteristics.Scientists can classify these organisms using molecular markers in combination with morphology.We successfully sequenced the complete nuclear ribosomal DNA.approximately 13 kb in length,from a marine Bangia population.We further analyzed the small subunit ribosomal DNA gene(nrSSU) and the internal transcribed spacer(ITS) sequence regions along with nine other marine,and two freshwater Bangia samples from China.Pairwise distances of the nrSSU and 5.8S ribosomal DNA gene sequences show the marine samples grouping together with low divergences(0-0.003;0-0.006,respectively) from each other,but high divergences(0.123-0.126;0.198,respectively) from freshwater samples.An exception is the marine sample collected from Weihai,which shows high divergence from both other marine samples(0.063-0.065;0.129,respectively) and the freshwater samples(0.097;0.120,respectively).A maximum likelihood phylogenetic tree based on a combined SSU-ITS dataset with maximum likelihood method shows the samples divided into three clades,with the two marine sample clades containing Bangia spp.from North America,Europe,Asia,and Australia;and one freshwater clade,containing Bangia atropurpurea from North America and China.

Phylogeny of Ptychostomum (Bryaceae,Musci) inferred from sequences of nuclear ribosomal DNA internal transcribed spacer (ITS) and chloroplast rps4

The phylogeny of Ptychostomum was first spacer （ITS） region of the nuclear ribosomal （nr） DNA DNA rps4 sequences. Maximum parsimony, maximum undertaken based on analysis of the internal transcribed and by combining data from nrDNA ITS and chloroplast likelihood, and Bayesian analyses all support the conclusion that the reinstated genus Ptychostomum is not monophyletic. Ptychostomum funkii （Schwagr.） J. R. Spence （≡ Bryum funkii Schwaigr.） is placed within a clade containing the type species of Bryum, B. argenteum Hedw. The remaining members of Ptychostomum investigated in the present study constitute another well-supported clade. The results are congruent with previous molecular analyses. On the basis of phylogenetic evidence, we agree with transferring B. amblyodon Mull. Hal. （≡ B. inclinatum （Brid.） Turton≡ Bryum archangelicum Bruch ＆ Schimp.）, Bryum lonchocaulon Mull. Hal., Bryum pallescens Schleich. ex Schwaigr., and Bryum pallens Sw. to Ptychostomum.

RNA-binding Protein UNR Promotes Glioma Cell Migration and Regulates the Expression of Ribosomal Protein L9

Objective To investigate the role of RNA binding protein—upstream-of-N-Ras(UNR)in the development of glioma and its molecular mechanism.Methods First,bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma.Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues.Next,UNR siRNAs were transfected in glioma cells,and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration.Then,the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq,RNA sequencing and ribosome profiling databases of human melanoma.RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells.Finally,western blot was used to detect the effect of UNR knockdown on ribosomal protein L9(RPL9)and RPL9 protein expression level in glioma cell lines.RPL9 siRNA was transfected in A172 and T98G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in highgrade glioma[GradeⅡ(n=126),GradeⅢ(n=51),GradeⅣ(n=128),P<0.001].UNR high expression levels were associated with poor prognosis(P=0.0177).UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples(P<0.01).Knockdown of UNR inhibited glioma cells migration(P<0.05),but did not inhibit glioma cells growth in three glioma cell lines.UNR binded the 3’untranslated region(UTR)of PTEN and RPL9 mRNAs.RPL9 protein was significantly highly expressed in most glioma cell lines(n=9)and knockdown of UNR resulted in a downregulation of RPL9 protein expression.

Characterization and analysis of ribosomal proteins in two marine calanoid copepods

Copepods are among the most abundant and successful metazoans in the marine ecosystem. However, genomic resources related to fundamental cellular processes are still limited in this particular group of crustaceans. Ribosomal proteins are the building blocks of ribosomes, the primary site for protein synthesis. In this study, we characterized and analyzed the c DNAs of cytoplasmic ribosomal proteins(c RPs) of two calanoid copepods, P seudodiaptomus poplesia and A cartia pacifi ca. We obtained 79 c RP c DNAs from P. poplesia and 67 from A. pacifi ca by c DNA library construction/sequencing and rapid amplifi cation of c DNA ends. Analysis of the nucleic acid composition showed that the copepod c RP-encoding genes had higher GC content in the protein-coding regions(CDSs) than in the untranslated regions(UTRs), and single nucleotide repeats(>3 repeats) were common, with "A" repeats being the most frequent, especially in the CDSs. The 3′-UTRs of the c RP genes were signifi cantly longer than the 5′-UTRs. Codon usage analysis showed that the third positions of the codons were dominated by C or G. The deduced amino acid sequences of the c RPs contained high proportions of positively charged residues and had high p I values. This is the fi rst report of a complete set of c RP-encoding genes from copepods. Our results shed light on the characteristics of c RPs in copepods, and provide fundamental data for further studies of protein synthesis in copepods. The copepod c RP information revealed in this study indicates that additional comparisons and analysis should be performed on different taxonomic categories such as orders and families.

Molecular Cloning,Genomic Organization and Functional Analysis of the Ribosomal Protein S30(RPS30) Gene from Arachis hypogaea

The ribosomal proteins are crucial for the maintenance of ribosomal translational efficiency and fidelity.In the study,we characterized the ribosomal protein S30(RPS30)gene from Arachis hypogaea that has been isolated through Genefishing analysis during defense responses to Ralstonia solanacearum.The cDNA of RPS 30 contained a 189 base pair(bp)open-reading frame encoding 62 amino acids.The genomic DNA consists of 272 bp containing two exons and one 83 bp intron.The RPS 30 mRNA transcript was mainly expressed in roots and leaves.The expression level of the RPS 30 mRNA transcripts was up-regulated sharply 6 h after bacterial challenge and was 12 times greater than that of the control group.The phylogenetic analysis for genes encoding proteins showed that RPS30 were conserved within dicotyledonous and monocotyledonous plants.d S extremely exceeded d N in all branches of the tree(d N/d S<1.0),indicating that functional constraint have acted on RPS 30 throughout evolution.

AtFKBP53 is a histone chaperone required for repression of ribosomal RNA gene expression in Arabidopsis

染色质结构充分为控制基因表示是重要的，但是位于染色质改变下面的机制不是理解。这里，我们报导 FKBP (FK506 有约束力的蛋白质) 打 immunophilin， AtFKBP53，拥有 histone 女伴活动并且为镇压被要求在 Arabidopsis 的 ribosomal 基因表示。AtFKBP53 蛋白质是有典型 peptidylprolyl isomerase (PPIase ) 的 multidomain FKBP 领域和几个高度控告的领域。用 nucleosome 汇编试金，我们证明 AtFKBP53 举办 histone 女伴活动，控告的酸的域为这项活动是足够的。我们证明 AtFKBP53 通过酸的领域与 histone H3 交往，而 PPIase 领域为 histone 女伴活动或 histone 绑定是非必需的。当 AtFKBP53 活动在 Arabidopsis 被减少或消除植物时， Ribosomal RNA 基因(18S rDNA ) 是 overexpressed。染色质 immunoprecipitation 试金证明 AtFKBP53 与 18S rDNA 基因染色质被联系，含有 AtFKBP53 镇压在染色质水平的 rRNA 基因。这研究在在染色质改变和抄写的规定工作的植物识别新 histone 女伴。

Nematode Diversity of Qingdao Coast Inferred from the 18S Ribosomal RNA Gene Sequence Analysis

The 18S ribosomal DNA gene (18S rDNA) sequences (approximately 1300 bp in length) were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coast in bulk with nematode spe- cific primers. The PCR products were cloned, re-amplified, digested with Rsa I and Hin6 I restriction endonucleases and separated in agarose gel. Among 17 restriction fragment length types, types 1, 2 and 6 covered 61.2%, 14.4% and 9.3% of the clones analyzed, respectively, while the remaining 14 only covered 21 clones, which accounted for 15.1% of the total. Twenty-four representative clones were sequenced and phylogenetically analyzed by referring to those currently available in RDP and GenBank databases. Al- though it was hard to assign these sequences to known species or genera due to the lack of the 18S rDNA sequence data of known marine free-living nematodes, the obtained sequences were assigned to the nematodes of Adenophorea. Among them, twelve se- quences were close to Pontonema vulgare and Adoncholaimus sp., four to Daptonema procerus and two (identical) to Enoplus brevis. Our results showed that free-living marine nematode diversities could be determined by PCR retrieving and analysis of the 18S rDNA sequences and an 18S rDNA sequence could be assigned to a species or a genus only if the 18S rDNA sequences of the free-living marine nematodes were accumulated to some extent.

18β-glycyrrhetinic acid regulates mitochondrial ribosomal protein L35-associated apoptosis signaling pathways to inhibit proliferation of gastric carcinoma cells

BACKGROUND Gastric carcinoma(GC)is a common gastrointestinal malignancy worldwide.Based on the cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the mechanism by which 18β-glycyrrhetinic acid(18β-GRA)regulates mitochondrial ribosomal protein L35(MRPL35)related signal proteins to inhibit the proliferation of GC cells.METHODS Cell counting kit-8 assay was used to detect the effects of 18β-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823.The apoptosis and cell cycle were assessed by flow cytometry.Cell invasion and migration were evaluated by Transwell assay,and cell scratch test was used to detect cell migration.Furthermore,a tumor model was established by hypodermic injection of 2.5×106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18β-GRA on GC cell proliferation,and quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect MRPL35 expression in the engrafted tumors in mice.We used the term tandem mass tag(TMT)labeling combined with liquid chromatography–tandem mass spectrometry to screen for differentially expressed proteins(DEPs)extracted from GC cells and control cells after 18β-GRA intervention.A detailed bioinformatics analysis of these DEPs was performed,including Gene Ontology annotation and enrichment analysis,Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis,and so on.Moreover,STRING database(https://string-db.org/)was used to predict proteinprotein interaction(PPI)relationships and Western blot was used to detect the expression of proteins of interest in GC cells.RESULTS The results indicated that 18β-GRA could inhibit the proliferation of GC cells in a dose-and timedependent manner.It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase.The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose,and the migration and invasiveness of GC cells were inhibited.The results of animal experiments showed that 18β-GRA could inhibit tumor formation in BALB/c nude mice,and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice.Using TMT technology,609 DEPs,among which 335 were up-regulated and 274 were down-regulated,were identified in 18β-GRA intervention compared with control.We found that the intervention of 18β-GRA in GC cells involved many important biological processes and signaling pathways,such as cellular processes,biological regulation,and TP53 signaling pathway.Notably,after the drug intervention,MRPL35 expression was significantly down-regulated(P=0.000247),TP53 expression was up-regulated(P=0.02676),and BCL2L1 was down-regulated(P=0.01699).Combined with the Retrieval of Interacting Genes/Proteins database,we analyzed the relationship between MRPL35,TP53,and BCL2L1 signaling proteins,and we found that COPS5,BAX,and BAD proteins can form a PPI network with MRPL35,TP53,and BCL2L1.Western blot analysis confirmed the intervention effect of 18β-GRA on GC cells,MRPL35,TP53,and BCL2L1 showed dose-dependent up/down-regulation,and the expression of COPS5,BAX,and BAD also increased/decreased with the change of 18β-GRA concentration.CONCLUSION 18β-GRA can inhibit the proliferation of GC cells by regulating MRPL35,COPS5,TP53,BCL2L1,BAX,and BAD.

Expression of the N-terminal 99 Amino Acids of Yeast Ribosomal Protein L3 in Transgenic Wheat Confers Resistance to Fusarium Head Blight

Fusarium head blight (FHB) is a worldwide disease for wheat or barley. The contamination of important agricultural products with the trichothecene mycotoxins such as deoxynivalenol (DON) or 4,15-diacetoxyscirpe-

Phylogenetic analysis of Pectinidae (Bivalvia) based on the ribosomal DNA internal transcribed spacer region

The ribosomal DNA internal transcribed spacer （ITS） region is a useful genomic region for understanding evolutionary and genetic relationships. In the current study, the molecular phylogenetic analysis of Pectinidae （ Mollusca： Bivalvia） was performed using the nucleotide sequences of the nuclear ITS region in nine species of this family. The sequences were obtained from the scallop species Argopecten irradians, Mizuhopecten yessoensis, Amusium pleuronectes and Mimachlamys nobilis, and compared with the published sequences of Aequipecten opercularis, Chlamys farreri, C. distorta, M. varia, Pecten maximus, and an outgroup species Perna viridis. The molecular phylogenetic tree was constructed by the neighbor-joining and maximum parsimony methods. Phylogenetic analysis based on ITS1, ITS2, or their combination always yielded trees of similar topology. The results support the morphological classifications of bivalve and are nearly consistent with classification of two subfamilies （Chlamydinae and Pectininae） formulated by Waller. However, A. irradians, together with A. opercularis made up of genera Amusium, evidences that they may belong to the subfamily Pectinidae. The data are incompatible with the conclusion of Waller who placed them in Chlamydinae by morphological characteristics. These results provide new insights into the evolutionary relationships among scallop species and contribute to the improvement of existing classification systems.

Involvement of 90-kuD ribosomal S6 kinase in collagen type Ⅰ expression in rat hepatic fibrosis

AIM: To investigate the relationship between 90-kuD ribosomal S6 kinase (p90RSK) and collagen type □expression during the development of hepatic fibrosis in vivo and in vitro.METHODS: Rat hepatic fibrosis was induced by intraperitoneal injection of dimethylnitrosamine. The protein expression and cell location of p90RSK and their relationship with collagen type □were determined by co-immunofluoresence and confocal microscopy. Subsequently, RNAi strategy was employed to silence p90RSK mRNA expression in HSC-T6, an activated hepatic stellate cell (HSC) line. The expression of collagen type □in HSC-T6 cells was assessed by Western blotting and real-time polymerase chain reaction. Furthermore, HSCs were transfected with expression vectors or RNAi constructs of p90RSK to increase or decrease the p90RSK expression, then collagen type □promoter activity in the transfected HSCs was examined by reporter assay. Lastly HSC-T6 cells transfected with p90RSK siRNA was treated with or without platelet-derived growth factor (PDGF)-BB at a final concentration of 20 μg/L and the cell growth was determined by MTS conversion.RESULTS: In fibrotic liver tissues, p90RSK was overexpressed in activated HSCs and had a significant positive correlation with collagen type □levels.In HSC-T6 cells transfected with RNAi targeted to p90RSK, the expression of collagen type □was down-regulated (61.8% in mRNA, P < 0.01, 89.1% in protein, P < 0.01). However, collagen type □promoter activity was not increased with over-expression of p90RSK and not decreased with low expression either, compared with controls in the same cell line (P = 0.076). Furthermore, p90RSK siRNA exerted the inhibition of HSC proliferation, and also abolished the effect of PDGF on the HSC proliferation.CONCLUSION: p90RSK is over-expressed in activated HSCs and involved in regulating the abnormal expression of collagen type □through initiating the proliferation of HSCs.

A phylogeny of the Passerida(Aves: Passeriformes)based on mitochondrial 12S ribosomal RNA gene

Background: Passerida is the largest avian radiation within the order Passeriformes. Current understanding of the high-level relationships within Passerida is based on DNA–DNA hybridizations; however, the phylogenetic relationships within this assemblage have been the subject of many debates.Methods: We analyzed the 12 S ribosomal RNA gene from 49 species of Passerida, representing 14 currently recognized families, to outline the phylogenetic relationships within this group.Results: Our results identified the monophyly of the three superfamilies in Passerida: Sylvioidea, Muscicapoidea and Passeroidea. However, current delimitation of some species is at variance with our phylogeny estimate. First, the Parus major, which had been placed as a distinct clade sister to Sylvioidea was identified as a member of the super family;second, the genus Regulus was united with the Sturnidae and nested in the Muscicapoidea clade instead of being a clade of Passerida.Conclusion: Our results were consistent with Johansson's study of the three superfamilies except for the al ocation of two families, Paridae and Regulidae.

Ribosomal proteins expression and phylogeny in alpaca (<i>Lama pacos</i>) skin

Ribosomal proteins (RP) has been reported as a central player in the translation system, and are required for the growth and maintenance of all cell kinds. RP genes form a family of homologous proteins that express in the stable pattern and were used for phylogenetic analysis. Here we constructed a cDNA library of alpaca skin and 13,800 clones were sequenced. In the cDNA library, RP genes from skin cDNA library of alpaca were identified. Then 8 RP genes were selected at random and built the phylogenetic trees from the DNA sequences by using parsimony or maximum likelihood methods for molecular and evolutionary analysis of ribosomal proteins. The results showed that the 42 RP genes of alpaca have been expressed in alpaca skin. They were highly conserved. The phylogeny inferred from all these methods suggested that the evolutionary distances of alpaca RP genes were closer to rat.

Cloning, purification and characterization of the ribosomal protein L11 from E. coli

A high-expression system of L11 was constructed and investigated its interaction with other elements of the ribosome using physicochemical methods. The gene rplK, coding for the protein L11 from the E. coli 50S ribosomal subunit was amplifyied, cloned and over-expressed. The protein L11 was purified under native and denaturing conditions, refolded and the structure of both proteins was compared. The protein L11 properly refolded from 6M urea after dialysis. Experiments on binding of proteins L11, RRF and EF-G from Escherichia coli were performed by ana-lytical centrifugation and Biacore. Specific binding between protein L11 and RRF by analytical cen-trifugation was not detected probably due to struc-tural reasons. These findings may be helpful in the design of new antibiotics that specifically disrupt the interactions in the “GTP-associated site” of the bac-terial ribosome, as many of them are not effective anymore. A common intrinsically disordered region of protein L11 was found to be the amino acid se-quence 86-97, while the residues 67-74, containing the linker region, are predicted to be disordered by DisEMBL.

Evaluation of Ribosomal RNA Removal Protocols for <i>Salmonella</i>RNA-Seq Projects

Next generation sequencing is a powerful technology whose application in sequencing entire RNA populations (RNA-Seq) of food-borne pathogens will provide valuable insights. A problem unique to prokaryotic RNA-Seq is removal of ribosomal RNA. Unlike eukaryotic messenger RNA (mRNA), bacterial mRNA species are devoid of polyadenylation at the 3’-end and thus the approach of affinity enrichment of mRNA using oligo-dT probes is not an option. Among several approaches to enriching mRNA molecules, removal of ribosomal RNA (rRNA) by subtractive hybridization has been widely used. This approach is a single-step procedure for which several rRNA-depletion kits are commercially available. We evaluated three commercially available rRNA-depletion kits to determine their respective efficiencies of rRNA removal from Salmonella enterica serovar Typhimurium strain SL1344. The three protocols achieved varying degrees of rRNA depletion and resulted in 8 to 1000-fold enrichment of mRNA. rRNA removal probes from two of the three kits were unable to titrate out 23S rRNA species while removal of 16S rRNA was less efficient. The Ribo-Zero kit was most efficient in eliminating 16S, 23S and 5S ribosomal RNA species from the transcriptome of S. enterica serovar Typhimurium strain SL1344.

Knockdown of 60S ribosomal protein L14‑2 reveals their potential regulatory roles to enhance drought and salt tolerance in cotton

Background:Cotton is a valuable economic crop and the main significant source of natural fiber for textile industries globally.The effects of drought and salt stress pose a challenge to strong fiber and large-scale production due to the ever-changing climatic conditions.However,plants have evolved a number of survival strategies,among them is the induction of various stress-responsive genes such as the ribosomal protein large(RPL)gene.The RPL gene families encode critical proteins,which alleviate the effects of drought and salt stress in plants.In this study,comprehensive and functional analysis of the cotton RPL genes was carried out under drought and salt stresses.Results:Based on the genome-wide evaluation,26,8,and 5 proteins containing the RPL14B domain were identified in Gossypium hirsutum,G.raimondii,and G.arboreum,respectively.Furthermore,through bioinformatics analysis,key cis-regulatory elements related to RPL14B genes were discovered.The Myb binding sites(MBS),abscisic acid-responsive element(ABRE),CAAT-box,TATA box,TGACG-motif,and CGTCA-motif responsive to methyl jasmonate,as well as the TCA-motif responsive to salicylic acid,were identified.Expression analysis revealed a key gene,Gh_D01G0234(RPL14B),with significantly higher induction levels was further evaluated through a reverse genetic approach.The knockdown of Gh_D01G0234(RPL14B)significantly affected the performance of cotton seedlings under drought/salt stress conditions,as evidenced by a substantial reduction in various morphological and physiological traits.Moreover,the level of the antioxidant enzyme was significantly reduced in VIGS-plants,while oxidant enzyme levels increased significantly,as demonstrated by the higher malondialdehyde concentration level.Conclusion:The results revealed the potential role of the RPL14B gene in promoting the induction of antioxidant enzymes,which are key in oxidizing the various oxidants.The key pathways need to be investigated and even as we exploit these genes in the developing of more stress-resilient cotton germplasms.