Co-expression of two chicken genes for purine biosynthesis from a bidirectional promoter
Abstract
GPAT and AIRC encode the enzymes that catalyze steps 1 and 6 plus 7, respectively, in the de novo purine biosynthesis. The avian genes were cloned and characterized and it was established that they are closely linked and transcribed divergently. The promoter, embedded in a CpG island, directs co-expression of GPAT and AIRC from distinct transcription start sites 229 bp apart. This is the first example of genes coding for structurally unrelated enzymes of the same pathway being arranged in this manner. This arrangement has the potential to provide for regulated co-expression comparable to that in a prokaryotic operon. In order to identify important cis-elements in the intergenic region, the promoter was scanned by deletion mutagenesis and a bireporter vector was used to assay transcriptional activity in both directions in transient transfection assays. The results show that the intergenic region is an integrated bidirectional promoter rather than a juxtaposition of two independent promoters. Removal of sequences containing the two Sp1 or CCAAT boxes on the AIRC side or the CCAAT box on the GPAT side reduced transcription significantly on both sides. Surprisingly, removal of $\sim$50 bases surrounding the AIRC transcription start site severely impaired transcriptional activity in both directions. Deletion of DNA sequences downstream of the AIRC transcriptional start site and around the GPAT transcriptional start site reduced activity in a side-specific manner. Gel retardation assays using HeLa nuclear extracts detected a specific complex formed on the presumed AIRC initiator element. Mobility shift competition assays limited the binding site to 41 bp around the AIRC transcription start and a 41 bp oligonucleotide with the same sequence retained specific binding. DNase I assays show that protein binding on this region induces a hypersensitive site on the coding strand, in front of the transcription start site, and methylation interference assays implicate nucleotides one turn of the helix downstream of the hypersensitive site in protein/DNA specific contacts. On the GPAT side, transfection data, gel retardation and DNase I footprint assays implicate an octamer-like motif in GPAT expression. The data suggest that the two genes share an integrated bidirectional promoter and that a novel Inr element plays a central role in coordinating expression of the divergently transcribed AIRC and GPAT genes.
Degree
Ph.D.
Advisors
Zalkin, Purdue University.
Subject Area
Biochemistry|Molecular biology
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