TY - JOUR
T1 - Functional characterization of the cis-regulatory elements of the rat ribophorin I gene
AU - Rajasekaran, Ayyappan K.
AU - Zhou, Zhongmin
AU - Prakash, Kulkarni
AU - Das, Gokul
AU - Kreibich, Gert
N1 - Funding Information:
We thank Dr David Sabatini for his encouragement and support and Dr Milton Adesnik for his advice. We gratefully acknowledge Dr Winship Herr for providing Oct-2 expression construct. We are grateful to Dr Diego Gravotta for helping us to quantitate the CAT results using Phosphorlmager. The help of Myrna Cort, Bernice Rosen, Heide Plesken and Jody Culkin in preparing the manuscript is gratefully acknowledged. Gokul Das is a fellow of Leukemia Society of America and a recipient of a Long Island Biological Association (LEBA) postdoctoral fellowship. This work was supported by a grant from the American Cancer Society (CD-514; GK), and by a grant from the National Institute of Health (GM 20277, Dr D. Sabatini).
PY - 1995/2/11
Y1 - 1995/2/11
N2 - The ribophorin I gene encodes a rough endoplasmic reticulum (RER) specific membrane protein which is a subunit of the oligosaccharyltransferase. To establish the functional activity of its promoter region we have performed transient gene transcription experiments employing plasmid constructs that contain 5′ flanking regions of the ribophorin I gene cloned upstream of the CAT reporter gene. Among the restriction fragments obtained from the 1.3-kilobase 5′ flanking region, a proximal fragment (-42 to +24) containing two GC-rich elements was required for basic promoter activity, while a fragment (-364 to +24) encoding an additional GC-box and an octamer like motif at -233 conferred the maximal promoter activity. In order to investigate the functionality of an octomer-like sequence co-transfection experiments were performed with Oct-2 cDNA and the CAT reporter gene containing the ribophorin I fragment (-364 to +24). A 3-4-fold increase in the transcriptional activity was observed with this construct. In addition, gel shift experiments showed Oct-2 binding to this construct. These results indicate that Oct-2 is most likely involved in the regulation of the ribophorin I gene transcription. We suggest that the GC-rich elements are necessary for constitutive ribophorin I expression while octamer motif binding proteins function synergistically with the GC-rich element binding proteins to increase the expression of the ribophorin I gene during the proliferation of RER.
AB - The ribophorin I gene encodes a rough endoplasmic reticulum (RER) specific membrane protein which is a subunit of the oligosaccharyltransferase. To establish the functional activity of its promoter region we have performed transient gene transcription experiments employing plasmid constructs that contain 5′ flanking regions of the ribophorin I gene cloned upstream of the CAT reporter gene. Among the restriction fragments obtained from the 1.3-kilobase 5′ flanking region, a proximal fragment (-42 to +24) containing two GC-rich elements was required for basic promoter activity, while a fragment (-364 to +24) encoding an additional GC-box and an octamer like motif at -233 conferred the maximal promoter activity. In order to investigate the functionality of an octomer-like sequence co-transfection experiments were performed with Oct-2 cDNA and the CAT reporter gene containing the ribophorin I fragment (-364 to +24). A 3-4-fold increase in the transcriptional activity was observed with this construct. In addition, gel shift experiments showed Oct-2 binding to this construct. These results indicate that Oct-2 is most likely involved in the regulation of the ribophorin I gene transcription. We suggest that the GC-rich elements are necessary for constitutive ribophorin I expression while octamer motif binding proteins function synergistically with the GC-rich element binding proteins to increase the expression of the ribophorin I gene during the proliferation of RER.
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U2 - 10.1093/nar/23.3.313
DO - 10.1093/nar/23.3.313
M3 - Article
C2 - 7885824
AN - SCOPUS:0028946137
SN - 0305-1048
VL - 23
SP - 313
EP - 319
JO - Nucleic acids research
JF - Nucleic acids research
IS - 3
ER -