Sequence requirements for transcriptional arrest in exon 1 of the murine adenosine deaminase gene.

Abstract

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.

Transcription elongation in the human c-myc gene is governed by overall transcription initiation levels in Xenopus oocytes.

“…Secondly, the Xenopus oocyte system has been used to study transcription elongation in a number of genes, including the human c-myc gene (3,54), mouse c-myc gene (3,45,59), human and mouse adenosine deaminase genes (8,9,40), and a-tubulin gene (34). The present study provides a word of caution about the use of the Xenopus transcription system in studies of transcription elongation.…”

“…This assay has been used to study transcription arrest from the human c-myc gene (3,33,54), the murine c-myc gene (45,59), the human and mouse adenosine deaminase genes (8,9,40), and the Xenopus oa-tubulin gene (34).…”

Regulation of eukaryotic gene expression by transcriptional attenuation.

“…Examples include c-fos (Collart et al, 1991;London et al, 1991;Mechti et al, 1991), c-myb (Bender et al, 1987;Watson, 1988), adenosine deaminase (ADA) (Chinsky et al, 1989;Lattier et al, 1989;Chen et al, 1990Chen et al, , 1991Ramamurthy et al, 1990), and tubulin (Middleton and Morgan, 1990). An additional site of premature transcriptional termination has recently been defined between the two promoters, P1 and P2, of the c-myc gene Meulia et al, 1992;.…”

Sequence requirements for transcriptional arrest in exon 1 of the human adenosine deaminase gene.

“…This type of control can occur very early after transcription initiation and, in some cases, has been documented to be under metabolic or developmental control (4,6,13,47). Premature termination of transcription has been found in many cellular genes, such as c-myc (6,16,26,39), L-myc (28), c-fos (18,30), c-myb (4,53), the human histone gene H3-3 (44), the adenosine deaminase (ADA) gene (8,9,31,33,41), the human epidermal growth factor receptor gene (19), the Drosophila hsp-70 gene (47), and several other Drosophila genes (48). In addition, control of transcription elongation is associated with the regulated expression of several viral transcription units, including those of simian virus 40 (20), adenovirus type 2 (34,38), polyomavirus (50), minute virus of mice (3,45), and human immunodeficiency virus (HIV) (25).…”

“…Previously, we have provided evidence that a transcriptional arrest site exists at the 5' end of murine and human ADA genes and that this arrest site is involved in the regulation of ADA gene expression (8,9,33,41 Northern blot analysis of RNA. Two micrograms of oocyte RNA per sample was separated by electrophoresis through 6% polyacrylamide-8 M urea denaturing gels and electroblotted onto nylon membranes (Zeta-Probe).…”