![]() Analysis of mRNA 3' end formation by modification interference: the only modifications which prevent processing lie in AAUAAA and the poly(A) site. Analysis in Cos-1 cells of processing and polyadenylation signals by using derivatives of the herpes simplex virus type 1 thymidine kinase gene. ![]() Citron B, Falck-Pedersen E, Salditt-Georgieff M, Darnell JE., Jr Transcription termination occurs within a 1000 base pair region downstream from the poly(A) site of the mouse beta-globin (major) gene.Nucleotide sequences in Xenopus 5S DNA required for transcription termination. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Polyomavirus late leader region serves an essential spacer function necessary for viability and late gene expression. Kinetics and efficiency of polyadenylation of late polyomavirus nuclear RNA: generation of oligomeric polyadenylated RNAs and their processing into mRNA. Efficiency of processing of viral RNA during the early and late phases of productive infection by polyoma virus. Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome. Links to PubMed are also available for Selected References. Get a printable copy (PDF file) of the complete article (1.6M), or click on a page image below to browse page by page. Full textįull text is available as a scanned copy of the original print version. These results also show that the multiple, spliced leaders on polyomavirus L-strand mRNAs, which arise as a result of inefficient termination and polyadenylation, are not necessary for efficient virus replication. These findings demonstrate that the rabbit beta-globin insert, which contains a strong polyadenylation signal, also contains at least part of a signal for termination of transcription by RNA polymerase II. Most importantly, termination of transcription by RNA polymerase II on ins 5 DNA was also increased compared with wild-type virus nearly 100% of polymerases terminated per traverse of the ins 5 genome. Furthermore, the presence of this efficient polyadenylation signal resulted in a 1.4- to 2.5-fold increase in the fraction of virus-specific RNAs that were polyadenylated. The beta-globin signal was efficiently recognized by the cleavage/polyadenylation machinery in mouse 3T6 cells infected with ins 5, signalling greater than 90% of the polyadenylation events on L-strand RNAs. Included in this fragment are all of the sequence elements required for efficient cleavage and polyadenylation of rabbit beta-globin RNA. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors.Ĭopyright (c) 2007 John Wiley & Sons, Ltd.We constructed a viable insertion mutant (ins 5) of polyomavirus which contains, upstream of the L-strand polyadenylation signal, a 94-nt fragment of rabbit beta-globin DNA. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors.
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