B.M.M. Dekker

Adenosine deaminase: characterization and expression of a gene with a remarkable promoter.

Cosmid clones containing the gene for human adenosine deaminase (ADA) were isolated. The gene is 32 kb long and split into 12 exons. The exact sizes and boundaries of the exon blocks including the transcription start sites were determined. The sequence upstream from this cap site lacks the TATA and CAAT boxes characteristic for eukaryotic promoters. Nevertheless, we have shown in a functional assay that a stretch of 135 bp immediately preceding the cap site has promoter activity. This 135‐bp DNA fragment is extremely rich in G/C residues (82%). It contains three inverted repeats that allow the formation of cruciform structures, a 10‐bp and a 16‐bp direct repeat and five G/C‐rich motifs (GGGCGGG) disposed in a strikingly symmetrical fashion. Some of these structural features were also found in the promoter region of other genes and we discuss their possible function. Knowledge of the exact positions of the intron‐exon boundaries allowed us to propose models for abnormal RNA processing that occurs in previously investigated ADA‐deficient cell lines.

One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity.

We have cloned and sequenced an adenosine deaminase (ADA) gene from a patient with severe combined immunodeficiency (SCID) caused by inherited ADA deficiency. Two point mutations were found, resulting in amino acid substitutions at positions 80 (Lys to Arg) and 304 (Leu to Arg) of the protein. Hybridization experiments with synthetic oligonucleotide probes showed that the determined mutations are present in both DNA and RNA from the ADA‐SCID patient. In addition, wild‐type sequences could be detected at the same positions, indicating a compound heterozygosity. Studies with ADA expression clones mutagenized in vitro showed that the mutation at position 304 is responsible for ADA inactivation.

Gene organization of the transforming region of adenovirus type 7 DNA

The sequence of the leftmost 11% of the weakly oncogenic human adenovirus type 7 (Ad7) DNA has been determined. This part of the Ad7 viral genome encompasses early region E1 which has been shown to be involved in the process of cell transformation in vitro (Dijkema et al., 1979). From the nucleotide sequence and determined coordinates of the E1 mRNAs, we are able to predict the primary structure of the polypeptides encoded by the transforming region of Ad7. The organization of the E1 region of Ad7 and of other adenovirus serotypes (Bos et al. 1981) leads to the proposal of a novel mechanism for gene regulation at the translational level in which protein synthesis can initiate at either the first or the second AUG triplet available in mRNA. The differences between the large E1b-specific tumor antigens of adenovirus types 12, 7 and 5 may explain the differences in oncogenicity of these viruses.

The nucleotide sequence of the leftmost XhoI fragment (6 %) of Simian adenovirus SA7P

The DNA of simian adenovirus SA7P was cloned in pBR322. The nucleotide sequences of the leftmost 2238 bp and the rightmost 188 bp of the viral genome were determined. SA7P DNA has an inverted terminal repeat of 183 bp. The sequence at the left terminus exhibits extensive homology with that of the E1 regions of human adenovirus 5, 7 and 12 DNAs. Based on this homology, the RNA coordinates and coding regions could be deduced. The sequenced SA7P DNA contains the entire E1A and part of the E1B region.

FUNCTIONAL ANALYSIS OF A REMARKABLE EUKARYOTIC PROMOTER, THE HUMAN ADENOSINE DEAMINASE PROMOTER: 14

We have cloned and characterized the human gene for adenosine deaminase (ADA)(ref.1). One surprising feature of this gene is the lack of the characteristic eukaryotic promoter elements (i.e. a TATA and a CAAT box) in the region (0-135) upstream of the cap site (+1). Nevertheless, this upstream region, which is extremely GC-rich (82%), was shown to have promoter activity in a transient expression assay (ref.1). It was hypothesized that the remarkable symmetrically disposed GC-rich motifs found in this promoter region play an important role in the regulation of the ADA gene. This was deduced from their striking homology with elements found in other genes (e.g. those for DHFR and PGK), especially with those elements in the SV-40 early promoter which have been proven to bind specific transcription factors (SP1) intimately (ref.2).To investigate the ADA promoter in more detail, we linked the promoter region to the chloramphenicol acetyl transferase (CAT) gene and determined its activity in cell lines derived from various tissues and representing different stages of differentiation. By in vitro mutagenesis of the ADA promoter and by competition experiments involving cotransfection with specific SV-40 early promoter sequences we are attempting to resolve the role of the GC-rich motifs in the functioning of this remarkable promoter. Ref.1: D.Valerio et al., The EMBO J.vol.4(2) 1985, in press.2: D.Gidoni et al., Nature 312: 409-413 (1984).