We have previously shown that a transcription arrest site near the 5' end of the murine adenosine deaminase (ADA) gene is significantly involved in the regulation of ADA gene expression. To facilitate the analysis of this transcription arrest site, we have analyzed the transcription products from cloned ADA gene fragments injected into Xenopus laevis oocytes. When genomic fragments spanning the 5' end of the ADA gene were injected into oocytes, a 96-nucleotide (nt) ADA RNA was the major transcription product. The 5' end of this RNA mapped to the transcription initiation site for the ADA gene, and its 3' terminus mapped 7 nt downstream of the translation initiation codon within exon 1. A 300-base-pair fragment of genomic DNA spanning the 5' end of the ADA gene was sufficient to generate the 96-nt transcript which accounted for approximately one-half of the transcription products from injected templates. Deletion of a segment of approximately 65 base pairs, located immediately downstream of the 3' terminus of the 96-nt transcript, resulted in a substantial reduction in the synthesis of the 96-nt transcript and a corresponding increase in the production of larger transcripts. These studies show that the transcriptional apparatus of X. laevis oocytes responds to the transcription arrest site associated with exon 1 of the murine ADA gene and that oocyte injections provide a convenient functional assay for additional mechanistic studies.

Analysis of human adenosine deaminase (ADA) gene transcription in four different cell lines indicated that a high density of RNA polymerase II complexes is present at the 5' end of the gene and that the extent of transcription elongation beyond the promoter-proximal region governs gene expression. To determine the sequence requirements for a potential transcription arrest site in the promoter-proximal region, genomic clones containing the ADA promoter, exon 1, and various lengths of intron 1 were injected into Xenopus laevis oocyte germinal vesicles. Transcription analysis indicated that nascent ADA transcripts were highly represented at the promoter-proximal region of the injected templates, suggesting that transcription arrest occurred in the oocyte transcription system. Analysis of the transcription products indicated that ADA transcription initiated at the authentic start site and that the most prominent, short ADA transcripts were 105 nucleotides in length. The 3' end of these transcripts mapped within exon 1, 10 nucleotides downstream of the translation initiation codon. Deletion analysis demonstrated that sequences within exon 1 were sufficient to specify the synthesis of the 105-nucleotide transcripts. Taken together, these data suggest that a transcription arrest mechanism operates in the promoter-proximal region of the human ADA gene and that regulation of elongation beyond this point plays a major role in regulating ADA gene expression.

A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplified copies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clones encompassing the entire ADA structural gene were isolated from this genomic library and used for subsequent structural and functional analyses. Nuclease protection and primer extension analyses served to identify the location of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity was found by functional analyses to reside within a 240-base-pair fragment which contains the transcription initiation sites. Sequences upstream of the transcription initiation sites are very G + C rich (77%) and include a 22 nucleotide stretch of deoxyguanylate residues and two potential Sp1 transcription factor-binding sites. Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that are highly conserved with regard to both sequence content and location and may represent genetic elements which are involved in ADA gene expression.