Nucleic Acids Research, 1994, Vol. 22, No. 12 2375-2382
© 1994
RNA |
Ribozyme mediated degradation of ß-amyloid peptide precursor mRNA in COS-7 cells
New York State Institute for Basic Research in Developmental Disabilities 1050 Forest Hill Road, Staten Island, NY 10314, USA
*To whom correspondence should be addressed
Received February 2, 1994. Revised May 6, 1994. Accepted May 6, 1994.
Two sets of eucaryotic expression vectors encoding trans-acting hammerhead ribozymes and frans-acting hairpin ribozymes were constructed. In one set of vectors ribozyme RNA transcription was placed under the control of a mouse mammary tumor virus long terminal repeat (MMTV-LTR). In the other set ribozyme expression was controlled by a metallothionein IIA (Mt-IIA) promoter. Each ribozyme was directed to the first target sequence in the Alzheimer amyloid peptide precursor mRNA (ßAPP mRNA), 5'
GUC3'. Ribozyme RNA transcribed from these vectors, which should cleave all six alternatively spliced forms of ßAPP mRNA as well as ßAPP pre-mRNA, was shown to cleave a ßAPP RNA substrate analog in vitro. Stably transfected COS-7 cell lines bearing both vector types were prepared. Steady-state levels of ßAPP mRNA were reduced 25–30% in cells containing either active or mutant hammerhead ribozyme vectors driven by the MMTV-LTR promoter grown in the presence of glucocorticoids. In cell lines bearing Mt-IIA driven ribozymes steady-state levels of ßAPP mRNA were reduced 67–80% in both hammerhead and hairpin ribozyme containing cell lines following promoter induction by glucocorticoids. These levels correlate with the appearance of low levels of induced ribozyme RNA. In contrast, steady-state 
-actin mRNA and G3PDH mRNA levels in these cells remained constant. Western blotting of cell extracts revealed that all forms of ßAPP were correspondingly reduced. Neither the RNA nor protein decreases observed in ribozyme transfected cell lines were observed in stably transfected control cells bearing the vector alone. These results suggest that ribozyme-mediated degradation of ßAPP mRNA in COS-7 cells does not depend on ribozyme cleavage.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Q. Zhang, D. W. Ohannesian, E. L. Kreklau, and L. C. Erickson Modulation of 1,3-Bis-(2-chloroethyl)-1-nitrosourea Resistance in Human Tumor Cells Using Hammerhead Ribozymes Designed to Degrade O6-Methylguanine DNA Methyltransferase mRNA J. Pharmacol. Exp. Ther., July 1, 2001; 298(1): 141 - 147. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. Fujita, T. Koguma, J. Ohkawa, K. Mori, T. Kohda, H. Kise, S. Nishikawa, M. Iwakura, and K. Taira Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli PNAS, January 21, 1997; 94(2): 391 - 396. [Abstract] [Full Text] [PDF]
|
|