1887

Chapter 81 : Modulation of Expression using an Antisense Strategy

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $15.00

Preview this chapter:
Zoom in
Zoomout

Modulation of Expression using an Antisense Strategy, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555815660/9781555813901_Chap81-1.gif /docserver/preview/fulltext/10.1128/9781555815660/9781555813901_Chap81-2.gif

Abstract:

Hsp60 mediates invasion of HeLa cells, blocks organelle trafficking, and is the most abundant protein secreted into the phagosomes of host cells throughout the course of intracellular multiplication. In the heat shock response is controlled by sigma factor 32 (RpoH) encoded by . Moreover, we do not know to what extent RpoH is either expressed or required for gene expression. In a study the authors focused on controlling levels of Hsp60 production by modulating expression of in order to evaluate the role of heat-shock protein (Hsp60) and the heat shock response in pathogenesis. The authors developed three strategies to alter expression: (i) construct an knockout mutant by allelic replacement; (ii) replace the endogenous promoter with an isopropyl- β-D-thio-galactopyranoside (IPTG)-inducible promoter; and (iii) to knock down levels of RpoH and HtpAB with antisense RNA. Either the gene is located in a dead zone for recombination or more likely the introduction of strong promoters into this locus causes constitutive expression of downstream genes associated with cell division (YEX). Preliminary data presented in a report in this chapter suggest that antisense can be used to knock down protein expression levels in .

Citation: Ewann F, S. Hoffman P. 2006. Modulation of Expression using an Antisense Strategy, p 336-338. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch81

Key Concept Ranking

Type IV Secretion Systems
0.46034512
0.46034512
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

Figures

Image of FIGURE 1
FIGURE 1

RNA level of in recombinant Philadelphia-1 strains. The recombinant strains containing the half length and half length or full length antisense constructs (lanes 2 to 5) were subjected to a 24-h induction with 5 mM IPTG. RNA was extracted and subjected to RT-PCR with primers specific for the (*) and (internal control) genes. The signal was compared to the one obtained for the parental strain containing either the empty vector (lane 1) or the same vector expressing (lane 6).

Citation: Ewann F, S. Hoffman P. 2006. Modulation of Expression using an Antisense Strategy, p 336-338. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch81
Permissions and Reprints Request Permissions
Download as Powerpoint
Image of FIGURE 2
FIGURE 2

Hsp60 production in the antisense-containing strains. strains Philadelphia-1 recombinant containing the empty vector (lane 1), the same vector expressing (lane 6), the half length, , and half length or full length antisense constructs (lanes 2 to 5) were subjected to a 24-h induction with 5mM IPTG. The protein extract obtained from the different strains were run on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis and transferred on a nitrocellulose membrane. After Ponceau Red staining (), the production of Hsp60 was assessed by immunoblotting with anti-Hsp60 antibodies ().

Citation: Ewann F, S. Hoffman P. 2006. Modulation of Expression using an Antisense Strategy, p 336-338. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch81
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555815660.ch81
1. Chien, M.,, I. Morozova,, S. Shi,, H. Sheng,, J. Chen,, S. M. Gomez,, G. Asamani,, K. Hill,, J. Nuara,, M. Feder,, J. Rineer,, J. J. Greenberg,, V. Steshenko,, S. H. Park,, B. Zhao,, E. Teplits-kaya,, J. R. Edwards,, S. Pampou,, A. Georg-hiou,, I. C. Chou,, W. Iannuccilli,, M. E. Ulz,, D. H. Kim,, A. Geringer-Sameth,, C. Golds-berry,, P. Morozov,, S. G. Fischer,, G. Segal,, X. Qu,, A. Rzhetsky,, P. Zhang,, E. Cayanis,, P. J. De Jong,, J. Ju,, S. Kalachikov,, H. A. Shuman, and, J. J. Russo. 2004. The genomic sequence of the accidental pathogen Legionella pneumophila. Science 305:19661968.
2. Cowing, D. W.,, J. C. Bardwell,, E. A. Craig,, C. Woolford,, R. W. Hendrix, and, C. A. Gross. 1985. Consensus sequence for Escherichia coli heat shock gene promoters. Proc. Natl. Acad. Sci. USA 82:26792683.
3. Garduno, R. A.,, G. Faulkner,, M. A. Trevors,, N. Vats, and, P. S. Hoffman. 1998. Immunolo-calization of Hsp60 in Legionella pneumophila. J. Bacteriol. 180:505513.
4. Garduño, R. A.,, E. Garduno, and, P. S. Hoffman. 1998. Surface-associated hsp60 chaperonin of Legionella pneumophila mediates invasion in a HeLa cell model. Infect. Immun. 66:46024610.
5. Morales, V. M.,, A. Backman, and, M. Bag-dasarian. 1991. A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. Gene 97:3947.
6. Weeratna, R.,, D. A. Stamler,, P. H. Edelstein,, M. Ripley,, T. Marrie,, D. Hoskin, and, P. S. Hoffman. 1994. Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60. Infect. Immun. 62:34543462.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error