Study On The Transcription Start Sites Of Genes In Brucella

Brucella species are typical zoonotic facultative intracellular pathogen,which could infect a variety of mammals,including sheep,cattle and pigs.They are mainly spread through skin mucosa,digestive tract,and respiratory tract.Brucellosis in livestock is characterized by abortion and infertility.Human infected with wave fever,arthritis,orchitis and other symptoms,and severe cases may even lose labor.Brucellosis has not only brought huge economic losses to the breeding industry,but also posed a great threat to public health.The capacity to survive and replicate in host cells is the central virulence characteristics of Brucella,but the potential mechanism has not yet been elucidated.Bacteria respond to a variety of environmental conditions by manipulating the expression of related genes to meet their own growth needs.Many regulatory factors function where RNA polymerase initiates messenger RNA synthesis.However,limited gene annotation is a current barrier to the study on gene expression regulation in Brucella.In this study,we present a method called Capping-RACE,which could be widely applied to the identification of transcriptional start sites in prokaryotes.Simultaneously,based on the principle of this method,we mapped the transcriptional start sites genome wide at single base resolution in Brucella with Pac Bio SMRT sequencing platform.Furthermore,we mutated the core gene that regulates the degradation of primary transcripts in bacteria.The detailed results are as follows:1.In this study,we proposed the concept and method of Capping-RACE.Primary transcripts were first treated with vaccinia capping enzyme to add a cap structure at the 5' ends.Following by reverse transcription,template-switching,nested PCR amplification,and then the products were cloned and sequenced to obtain the transcription start sites of genes.2.We verified the feasibility of Capping-RACE by the template-switching experiment with in vitro transcribed RNA.And then this method was confrmed to be sequence independent using the in vitro transcribed RNAs with different 5' terminal sequences and structures.Using this new technique,we successfully identifed the transcription start sites of protein-coding genes and non-coding genes in Escherichia coli and Brucella melitensis.To ascertain the sensitivity of Capping-RACE,we used a variety of starting input of total RNA to identify the transcription start site of omp A.The result showed that Capping-RACE only need 1 pg of RNA to determine the gene transcription start site.It is more sensitive than RLM-RACE.3.Combining the principle of Capping-RACE and Pac Bio SMRT sequencing platform,we developed Capping-seq strategy for genome-Scale mapping of transcription start sites in Brucella.The results of q PCR and high throughput sequencing showed that RNase H digestion method could effectively remove r RNA from total RNA samples.We identified 2367 transcription start sites and 309 potential transcription termination sites in Brucella melitensis 16 M strain by Capping-seq.4.The 5' ends analysis of the Brucella transcripts revealed that the majority of transcripts initiated with a purine nucleotide.303 genes were associated with two or more transcription start sites.260 transcripts contained 5' UTRs longer than 100 nt.These results indicated that there may be a complex regulatory network at the 5' ends of transcripts in Brucella.The promoter analysis results suggested that gene transcription region contained-35 and-10 elements with consensus sequences of TTGNNN and TATNNN,respectively.5.We constructed the rpp H deletion mutant strains of E.coli and B.melitensis,and confirmed that rpp H is involved in the regulation of transcripts degradation.In conclusion,we present the principles and methods of Capping-RACE and Capping-seq.The strategies provide tools for the study on transcriptional initiation,gene regulation,environmental stress and pathogenic mechanism in bacteria,such as Brucella and E.coli.