Cloning and Phylogenetic Analysis of RNA Helicase p68 Encoding DDX5 Gene from Human MCF7 Cell Line.
In this study genomic DNA was isolated from MCF7 cells and used as template for polymerase chain reaction (PCR) to amplify p68 by using sequence specific primers. The PCR product was purified ligated into pMSCV-GFP vector and transformed into E. coli DH5a competent cells. Restriction analysis was performed to confirm the cloning. Further the gene was sequenced and on the basis of this partial sequence a phylogenetic tree was made to access homology among different organisms. In present study gene encoding p68 in human was found to be more close to gene encoding p68 in Sus scrofa and Pan troglodytes. The results obtained from present study will be helpful to study the role of p68 in regulation of gene expression in humans in future. Copyright 2014 Friends Science Publishers.
Keywords: p68; RNA helicase; phylogeny; MCF7; Cloning.
RNA helicases is a big group of conserved proteins present in all groups of organisms. Cell development cell division cell differentiation mRNA synthesis and processing assembly of ribosome and protein synthesis are different cellular function affected by these enzymes. These are important for remodeling of ribonucleoproteins as they have the ability to disrupt interactions between protein and RNA (Tanner and Linder 2001; Zhu and Ren 2012).DEAD-box (DDX) proteins comprising of 38 members in humans is the biggest group of RNA helicases; presence of signature amino acid sequence (Asp-Glu-Ala- Asp or D-E-A-D) which is highly conserved is the reason for this name. Manipulation of RNA structures is a vital role played by many proteins of DEAD box family. The presence of RNA either double-or single-stranded stimulates ATP hydrolysis which is carried out by these DDX proteins but in a few processes these DDX proteins act as true RNA helicase. DEAD box proteins act as ATP-dependent helicases they disrupt interactions in RNAprotein complexes which are involved in vital processeslike degradation of RNA transcription mRNA processing translation RNA export from nucleus (Linder 2006). Dysregulated expression and genomic amplification of RNA helicases (DEAD box) has implications in the development of cancer. Many of these proteins perform important functions in processes related to transformation or cellular proliferation. DEAD box proteins are the subject of interest in cancer research due to their roles in progression or development of cancer (Godbout et al. 2007; Schroder 2010).p68 (DDX5) is a prototypic member of the DEAD box family (Ford et al. 1988) and is an established ATPase and RNA helicase (Hirling et al. 1989; Iggo and Lane1989). Previous reports have shown that p68 expression is growth and developmentally regulated and that p68 is overexpressed and abnormally polyubiquitylated incolorectal tumours (Stevenson et al. 1998; Causevic et al.2001). In vitro experiment has shown p68 to be essential for splicing of pre-mRNA (Liu 2002) and it participates in regulation of alternative splicing of c-H-ras (Guil et al.2003). It predominates in the cell nucleus due to its role in transcriptional regulation as well as pre-mRNA splicing. It is clear now that p68 moves between cytoplasm and nucleus as it also stays in cytoplasm for a short period of time. This movement between nucleus and cytoplasm takes place via RanGTPase-dependent pathway with the help of two nuclear localizaton and two nuclear exporting signal sequences (Wang et al. 2009; Mustafa et al. 2013).Presence of p68 in all groups of living organisms has been reported previously but little is known about its evolution. Any knowledge about the evolution of this gene would be helpful to establish its role in other organisms as well. Therefore present project was designed to isolate and clone DDX5 gene from human and to study its phylogenetic relation with different organisms.
Materials and Methods
Maintenance of Cell Culture
MCF7 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM glutMAx) supplemented with 10% Fetal Bovine Serum (FBS) at 37oC in 5% CO2 atmosphere (Jawaid et al. 2010). All culture reagents were obtained from Gibco Lifesciences USA.
Isolation and Manipulation of DNA
Genomic DNA was isolated from MCF7 cells by usingQIAamp(R) DNA Micro kit from Qiagen and visualized on1% agarose gel.
PCR Amplification of p68 Gene
Oligonucleotides used for amplification were:sense BglII; 5` GGAAGATCT ACCAT GTCGGGTTATTCGAGTGACantisense XhoI: 5` CCGCTCGAG TT AAGCGTAGTCTGGGACGTCGTATGGGTAAmplification was performed by using GoTaq(R) Hot Start Polymerase from Promega by following the manufacturer's protocol. Initial denaturation was performedat 94oC for 30 s 38 cycles (94oC 30 s 59oC 30 s 72oC 1min 40 s) with final extension at 72oC for 10 min. PCR amplification of the target sequence was confirmed by agarose gel electrophoresis. Digested PCR product was purified by using the QIAquick PCR Purification Kit anddigested vector was purified by using QIAquick GelExtraction Kit from Qiagen.
Cloning of p68 Gene
The ligation of purified PCR p68 sequence was performed by using T4 DNA ligase from Fermantas. DH5a competent cells from Invitrogen were used for transformation of recombinant vectors. Few bacterial colonies obtained after transformation were chosen to select positive clone by plasmid preparation. Colonies were grown overnight in LB medium and used for miniprep analysis by PureYield Plasmid Miniprep System (Promega). Ampicillin was used at concentration of 100 mg/mL. Purified recombinant vectors were double digested with respective enzymes from Fermentas by using standard buffering conditions at 37oC for 1 h and 30 min to confirm the presence of insert.
Sequencing and Homology
After restriction analysis positive clones were sequenced from MillGEN France. The sequence alignment between clone and known DDX5 sequence was performed by using CLUSTAL W software. BLAST (Basic Local Alignment Search Tool) was used to find similarity in sequence of DDX5 in different organism. The nucleotide sequence obtained after sequencing corresponding to the gene encoding p68 was used to make Neighbor joining Pylogenetic tree by using MEGA5 software (Yu et al.2012).
PCR Amplification and Cloning of the Gene MCF7 cells were cultured and maintained in DMEM for isolation of genomic DNA. Isolation of genomic DNA was confirmed by agarose gel electrophoresis. p68 was amplified by PCR using DNA template and amplification was confirmed by agarose gel electrophoresis (Fig. 1). Concentration of the PCR product (ng/L) was 357.
After PCR amplification; amplified product and vector were double digested with restriction enzymes and purified. After purification p68 was cloned into pMSCV-MIGR1- IRES-GFP vector and confirmed after miniprep analysis. Separation of p68 gene from the vector (Fig. 2) confirmed cloning of the gene.
BLAST (Basic Local Alignment Search Tool) has shown that this partial p68 gene sequence is 99% similar with Pan troglodytes DDX5 mRNA (AK306198.1) 94% similarity with DEAD box polypeptide 5 (DDX5) mRNA (NM_001191395.1) of Bos taurus and DDX5 mRNA (NM_001007613.1) of Rattus norvegicus was 93% similar.
Successful cloning produced p68 clone which can be expressed and purified for further study of its role in regulation of gene expression in different human diseases.
To study the genetic evolution of the human DDX5 a phylogenetic tree (Fig. 3) was constructed from the nucleotide sequences retrieved through BLAST (Basic Local Alignment Search Tool). We used neighbor joining method (NJ) to construct the tree. It calculates pairwise distance between sequences and form groups of sequences that are similar. It is computationally simple therefore speedy method (Harrison and Langdale 2006). Phylogenetic analysis has shown that the deadbox p68 (DDX5) of Human sapiens Sus scrofa and Pan troglodytes are found at equal distance therefore they are evolutionary close. While DDX5 of Rattus norvegicus and Macaca mulatta are more divergent from ancestor gene.
It is suggested that DDX5 of Human sapiens and of Sus scrofa and Pan troglodytes have originated from same ancestors.
In present study approximately 2 kb fragment of p68 was cloned from human. Previously RAlssler et al. (2000) cloned p68 from human to find its structural organization and found that p68 consisted of 13 exons and 12 introns. Northern blot revealed two forms of p68 mRNA; 2.3 kb fragment corresponding mature mRNA and 4.4 kb fragment representing alternatively or incomplete spliced mRNA.
Information that p68 is encoded by a single gene locus in humans came from Iggo et al. (1989). Clone prepared during the study can be expressed to study its function inhuman cells as other researchers have also cloned this gene to study its function. RNA helicases help in transcription either by stabilizing primary transcripts or its release from the template after its completion (Eisen and Lucchesi 1998). RNA helicase p68 was first identified as 65 kDa protein (p65) by methylene blue-mediated cross-linking (MB-cross- linked) interacting with the U1snRNP when it was bound at5`ss of intron in spliceosome formation by Liu et al. (1998). Later on Liu (2002) used monoclonal antibody PAb204 for immunoprecipitation of HeLa nuclear extract it precipitated p68. Same antibody also precipitated MB-cross-linked p65. MB-cross-linked and immunoprecipitation with polyclonal antibody PAbN1 (against p68) again precipitated p65 which confirmed that p65 was identical to RNA helicase p68. p68 cross links to 5`ss of RNA component of U1snRNP and is essential for in vitro splicing (Liu et al. 1998; Liu 2002). Alternative splicing of H-Ras is modulated by p68 production of oncogenic p21 H-Ras enhanced in the presence of p68 as it inhibit the inclusion of IDX (Guil et al.2003; Camats et al. 2008). Considering this activity p68 promotes tumor formation by promoting oncogenic p21 H- Ras production. p68 is not only involved in development regulation in humans but in other organisms as well.The phylogenetic study of this highly conserved p68 encoding gene from Homo sapiens has shown its presence in diverse groups of organisms. Both p68 and p72 proteins are involved in cell proliferation/transformation and cellular development in humans (Fuller-Pace 2006). Recently Fang et al. (2011) identified DDX5 homolog (Cq-DDX5) in Cherax quadricarinatus (fresh water crayfish) where this homolog was found to play an important role in early ontogenesis and spermatogenesis.In conclusion we have successfully isolated and cloned p68 gene from MCF7 cells. This gene is overexpressed in many of human cancers therefore it would be of great interest to overexpress the protein and study how overexpression is involved in the development of cancer Which level of gene expression regulation is affected byoverexpression The comparison of DDX5 gene from different organisms would be useful for detailed phylogenetic analysis and to establish its role in those organisms.
We are thankful to Dr. Stephan Vagenr INSERM U981 France for providing the facilities to conduct this research work.
Camats M. S. Guil M. Kokolo and M. Bach-Elias 2008. P68 RNA helicase (DDX5) alters activity of cis- and trans-acting factors of the alternative splicing of H-Ras. PLoS One 3 E2926Causevic M. R.G. Hislop and N.M. Kernohan 2001. Overexpression and poly-ubiquitylation of the DEAD-box RNA helicase p68 in colorectal tumours. Oncogene 20: 77347743Eisen A. and J.C. Lucchesi 1998. Unraveling the role of helicases intranscription. Bioessays 20: 634641Fang D.A. Q. Wang J. Wang L. He L.H. Liu and Y. Wang 2011. A novel DDX5 gene in the freshwater crayfish Cherax quadricarinatus is highly expressed during ontogenesis and spermatogenesis. Genet. Mol. Res. 10: 39633975Ford M.J. I.A. Anton and D.P. Lane 1988. Nuclear protein with sequencehomology to translation initiation factor eIF-4A. Nature 332: 736738Fuller-Pace F.P. 2006. DExD/H box RNA helicases: multifunctional proteins with important roles in transcriptional regulation. Nucl. Acids Res. 34: 4206-15Godbout R. L. Li Z.R. Liu and K. Roy 2007. Role of DEAD box 1 in retinoblastoma and neuroblastoma. Future Oncol. 3: 575587Guil S. R. Gattoni M. Carrascal J. Abian J. Stevenin and M. Bach-Elias2003. Roles of hnRNP A1 SR proteins and p68 helicase in c-H-ras alternative splicing regulation. Mol. Cell. Biol. 23: 29272941Harrison C.J. and J.A. Langdale 2006. A step by step guide to phylogenyreconstruction. Plant J. 45: 561572Hirling H. M. Scheffner T. Restle and H. Stahl 1989. RNA helicaseactivity associated with the human p68 protein. Nature 339: 562564Iggo R.D. and D.P. Lane 1989. Nuclear protein p68 is an RNA-dependentATPase. EMBO J. 8: 1827Iggo R. A. Gough W. Xu D.P. Lane and N.K. Spurr 1989.Chromosome mapping of the human gene encoding the 68-kDa nuclear antigen (p68) by using the polymerase chain reaction. Proc. Natl Acad. Sci. USA 86: 62116214Jawaid K. S.R. Crane J.L. Nowers M. Lacey and S.A. Whitehead 2010.Long-term genistein treatment of MCF-7 cells decreases acetylated histone3 expression and alters growth responses to mitogens and histone deacetylase inhibitors. J. Steroid. Biochem. Mol. Biol. 120: 164171Linder P. 2006. DEAD-box proteins: a family affair active and passiveplayers in RNP-remodeling. Nucl. Acids Res. 34: 41684180Liu Z.R. B. Sargueil and C.W. Smith 1998. Detection of a novel ATPdependent cross-linked protein at the 5` splice site U1 small nuclear RNA duplex by methylene blue-mediated photo-cross-linking. Mol. Cell. Biol. 18: 69106920Liu Z.R. 2002. p68 RNA helicase is an essential human splicing factor that acts at the U1 snRNA-5' splice site duplex. Mol. Cell. Biol. 22:54435450Mustafa R. J.J. Qi. X.L. Ba Y.Y. Chen C.M. Hu X.L. Liu L.L. Tu Q.J.Peng H.C. Chen and A.Z. Guo 2013. In vitro quinolones susceptibility analysis of chinese mycoplasma bovis isolates and their phylogenetic scenarios based upon QRDRs of DNA topoisomerases revealing a unique transition in ParC. Pak. Vet. J.33: 364369RAlssler O.G. P. Hloch N. SchA1/4tz T. Weitzenegger and H. Stahl 2000.Structure and expression of the human p68 RNA helicase gene.Nucl. Acids Res. 28: 932939Schroder M. 2010. Human DEAD-box protein 3 has multiple functions ingene regulation and cell cycle control and is a prime target for viral manipulation. Biochem. Pharmacol. 79: 297306Stevenson R.J. S.J. Hamilton D.E. MacCallum P.A. Hall and F.V.Fuller-Pace 1998. Expression of the dead box' RNA helicase p68 is developmentally and growth regulated and correlates with organ differentiation/maturation in the fetus. J. Pathol. 184: 351359Tanner N.K. and P. Linder 2001. DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol. Cell. 8: 251262Wang H. X. Gao Y. Huang J. Yang and Z.R. Liu 2009. p68 RNAhelicase is a nucleocytoplasmic shuttling protein. Cell Res. 19:13881400Yu L. L. Zhang L. Sun J. Lu W. Wu C. Li Q. Zhang F. Zhang C. JinX. Wang Z. Bi D. Li and M. Liang 2012. Critical epitopes in the nucleocapsid protein of SFTS virus recognized by a panel of SFTS patients derived human monoclonal antibodies. PLoS ONE. 7: 110Zhu W. and X. Ren 2012. Isolation genome phylogenetic analysis and in vitro rescue of a newly emerging porcine Circovirus Type 2. Pak. Vet. J. 32: 165170
|Printer friendly Cite/link Email Feedback|
|Publication:||International Journal of Agriculture and Biology|
|Date:||Jun 30, 2014|
|Previous Article:||Addition to the Boletes: Boletus pakistanicus sp. nov. from the Coniferous Forests of Pakistan.|
|Next Article:||Developmental Duration and Predatory Efficiency of Episyrphus balteatus on Four Aphid Species in Pakistan.|