Abstract
The first step in the assembly of the dolichol-linked oligosaccharides required for asparagine-linked glycosylation in eukaryotes is catalyzed by a tunicamycin-sensitive, dolichol phosphate-dependent N-acetylglucosamine-1-phosphate transferase (GPT). A fragment of the gene encoding the enzyme from Chinese hamster ovary (CHO) cells was partially cloned and characterized by a novel strategy. By stepwise selection, CHO cells were made 80-fold resistant to tunicamycin and found to have 10-fold elevated levels of GPT activity. Using a cloned segment of the yeast ALG-7 gene, which encodes the putative GPT from yeast, an amplified gene was identified by Southern blotting of the CHO DNA and a 6.6-kilobase segment of the gene was molecularly cloned. A family of RNA molecules in the 2.0-2.2-kilobase range identified with a probe from this gene was overexpressed in the resistant cells. The cloned DNA revealed a 24-amino acid residue sequence that was 92% conserved with the corresponding yeast sequence.
Highlights
The first step in the assembly of the dolichol-linked GlcNAc-P-P-Dol. It has been the subject of extensive study oligosaccharides required for asparagine-linked gly- because it is a possible control point for asparagine-linked cosylation in eukaryotes is catalyzed by a tunicamycin- glycosylation andthus maybe regulated by a number of sensitive, dolichol phosphate-dependent N-acetylglu- molecules such as phospholipids ( 2, 3 ) and dolichol sugars (4, cosamine-1-phosphate transferase (GPT)
In order to study further GPT, we have employed a novel strategy to amplify and identify the putative structural gene encodes the putative GPT from yeast,an amplified for GPT in Chinese hamster ovary (CHO) cells
The work described here extends theoriginal observations made with Tn-resistant CHO cells (9) and demonstrates amplification and cloning of a genomic fragment that is homologous to the putative yeast GPT structuralgene
Summary
A fragment of the gene encoding the enzyme from Chinese hamster ovary (CHO) cells waspartially cloned and characterized by a novel strategy. In order to study further GPT, we have employed a novel strategy to amplify and identify the putative structural gene encodes the putative GPT from yeast,an amplified for GPT in Chinese hamster ovary (CHO) cells. The work described here extends theoriginal observations made with Tn-resistant CHO cells (9) and demonstrates amplification and cloning of a genomic fragment that is homologous to the putative yeast GPT structuralgene. After incubation for 16 h at 42 "C, the filter was resistant cells were plated in 100-mm dishes and harvested after 4 washed twice for 15 min at 50 "C with 0.5% bovine serum albumin days of growth.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
