M.G.C. Duyvesteyn

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.

Cloning of human adenosine deaminase cDNA and expression in mouse cells

A previously isolated partial cDNA sequence encoding human adenosine deaminase (ADA) was used to probe a cDNA library prepared from human cultured cell mRNA. Clones containing a combined overlapping length of 1462 bp were isolated and sequenced. One of these was found to include the entire ADA coding region. An open reading frame consisting of 363 codons was identified, predicting a polypeptide of Mr 40762. A mammalian expression plasmid was constructed, positioning the ADA coding sequence to be under transcriptional control of the mouse metallothionein promoter. Transfection of cultured mouse L-cells with this plasmid resulted in the acute expression of human ADA enzymatic activity, as assayed by isoelectric focusing.

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.

Isolation of cDNA clones for human adenosine deaminase

Clones encoding human adenosine deaminase (ADA) were isolated from a cDNA library made from the lymphoblastoid cell line MOLT-4. The isolation procedure was based on the selection of clones hybridizing with a radioactive probe complementary to an RNA preparation, which had been highly enriched in ADA-specific mRNA. The latter RNA preparation was obtained by size-fractionating MOLT-4 RNA and selecting fractions that were translatable into ADA. The assay for the presence of ADA in the in vitro translation products, was based on immunoprecipitation with a specific anti-ADA serum. The antiserum used was shown to precipitate a 42-kDal protein with the properties of ADA. Positive clones were further screened by means of hybrid-released in vitro translation assays. Two clones were obtained which were able to select mRNA that could be translated into a 42-kDal protein immunoprecipitable with the ADA-antiserum. By use of Southern blots containing DNA from somatic cell hybrids, one of these ADA cDNA clones was assigned to the human chromosome 20 known to contain the ADA gene.

Sequence-specific endonucleases in strains of Anabaena and Nostoc

The complements of restriction endonucleases of 12 strains of cyanobacteria were determined in cell-free extracts, and were compared with the complements of restriction activities assessed by measuring the relative efficiencies of plating of cyanophages on those cyanobacteria. The hosts which were susceptible to all of the phages contained endo R · AvaI and endo R · AvaII, and in several cases probably endo R · AvaIII, or isoschizomers of these enzymes. Three hosts which were lysed by only a subset (1 or 3) of the phages contained different restriction endonuclease. Anabaena sp. PCC 7120 showed apparent phenotypic restriction of phage An-22 grown in hosts with (isoschizomers of) AvaI, II and III, but no corresponding endonuclease has yet been detected in vitro. Nostoc sp. ATCC 29131 (PCC 6705) was found to contain a restriction enzyme, NspBII, with hitherot unknown specificity, C(A/C)GC(T/G)G.

Introduction of sequences encoding functional human adenosine deaminase into mouse cells using a retroviral shuttle system

A retroviral packaging system was used to generate a murine virus carrying sequences encoding human adenosine deaminase (ADA). To this end, human ADA cDNA was inserted into the retroviral shuttle vector pZIP-NeoSV(X)1. This vector provides all of the cis-acting sequences necessary for the efficient packaging and transmission of the viral genome as well as a selectable gene for G418 resistance. Transfection of this recombinant plasmid into cells that provide essential virus products (psi-2 cells) yielded cell lines that stably produced virions carrying the coding sequence of human ADA. We have used these virions to infect NIH3T3 cells, which after 48 h synthesized catalytically active human ADA. Furthermore, G418-resistant cell lines were obtained from the virus-infected NIH3T3 cells that stably produced the human ADA enzyme.

Are sequence-specific deoxyribonucleases of value as taxonomic markers of cyanobacterial species?

Three nucleotide sequence-specific deoxyribonucleases present in extracts of Anabaena subcylindrica have been purified and characterized. Endo R·AsuI recognizes and cleaves the nucleotide sequence G↓GNCC (Hughes et al., 1980) while Endo R·AsuII and III split the sequences TT·CGAA and GPu↓CGPyC, respectively (this paper). An Anabaena strain “Waterbury” converging genetically at the 30–35% level with both A. subcylindrica and A. cylindrica (as judged by DNA-DNA hybridization, in vitro) was shown to possess the endonuclease pattern typical for A. cylindrica (de Waard et al., 1978). The usefulness of these specific endonucleases as taxonomic markers for the classification of cyanobacteria is discussed.

Two sequence-specific endonucleases from Anabaena oscillarioides

There has been an increasing number of reports on sequence-specific endodeoxyribonucleases (endo R.) in cyanobacteria (blue-green algae) [l-4]. As their cleavage specificities have proven to be different from those of the many bacterial restriction enzymes already characterized, these new enzymes have been useful additions to the ever expanding endo R. catalog. We report here the isolation of two such endonucleases from one strain of Anabaena oscillarioides (Am1 and II) which cleave the nucleotide sequences 5’ TGCjGCA 3’ and 5’ GPuJCGPyC 3’, respectively.

A new sequence-specific endonuclease from a thermophilic cyanobacterium, mastigocladus laminosus

The strain used (CCAP 1447/l) was obtained from the Culture Centre of Algae and Protozoa (Downing St, Cambridge) and originated from hot springs in New Zealand. It was grown in aqueous medium D [9] containing (mg/l): nitrilotriacetate, 100; Na2HP04, 1 10;NaN03,700;KN03, 100;NaC1,8;MgS04. 7H20, 100; CaCl*, 50; FeC13 complexed with EDTA, 0.3 and trace amounts of MnZC, Zn2+, Cu’+, Co’+, MOO:and BOi[5] buffered with 10 mM 2-[4-(2-hydroxyethyl)-piperazinyl( I)] ethane sulfonate (Hepes) at pH 8.1. A 1500 ml cylindrical culture vessel containing this medium was placed in a waterbath at 45°C and sterile air was blown in from below. Illumination was done by 2 fluorescent lamps at 40 cm distance (-1500 Lux). The inoculum was grown in 3 large (14 cm diam.) Petri dishes with medium solidified with 1.5% agar at 37°C. It took 3 weeks to prepare 10 g cells/l liquid culture from this inoculum. The various materials and methods used for determining the properties of the cleavage site of the Mastigocladus 1aminosusMla I enzyme were as in [5,6].

Isolation and characterization of a sequence-specific deoxyriboendonuclease from calothrix scopulorum

Sequence-specific deoxyriboendonucleases have been isolated from bacteria and fungi [ 6,8]. Except in a few cases the nucleotide sequences recognized by the cyanobacterial enzymes have been shown to be unique. In this report we describe the isolation and characterization of an endonuclease (endo R.CscI) from a cyanobacterium, Calothrix sco~ulorum which cleaves the deoxynucleotide sequence CCGC ~GG. Isoschizomers have been found previously in a fungus and in two bacterial strain~ 6]. The strain used (CCAP 1410/5) obtained from the Culture Centre of Algae and Protozoa (Downing St., Cambridge U,K.) was grown on medium BG-II [ 5] with aeration (95% air, 5% CO 2) at 28°C with illumination from two fluorescent lamps (i000 Lux). The algal material harvested from the culture did not reveal the presence of bacteria when viewed in the microscope after staining. Twenty grams of wet cells were freeze-thawed three times immediately prior to processing. The initial phase of the purification procedure of the enzyme was as described [i]. The dialyzed crude enzyme was then chromatographed on a 20 x 2.5 cm column of Whatman PII phosphocellulose using a 400 ml gradient of 0 to 0.6 NaCI in 20 mM potassium phosphate buffer, pH 7.4, containing 2 mM 2-mercaptoethanol and 10% glycerol; it eluted at around 0.5 M NaCI. Subsequent purification of the endonuclease was on a 5 x 1 cm Whatman DE 52 column with a 40 ml gradient of 0 to 0.4 M KCI in 10 mM Tris HCI buffer, pH 7.4 containing 2 mM 2-mercaptoethanol and 10% glycerol. The enzyme (eluting midway) was assayed by its cleaving activity towards 4 sites on bacteriophage ~ DNA as judged by agarose gel electrophoresis; it appeared free from other nucleolytic activities. The specificity of cleavage of the enzyme was established using a DNA molecule of known structure which was composed of a 5789 bp long Xho-C fragment of adenovirus 5 (leftmost 16%) contained in the vector plasmid pAT 153([ 7] and R. Bernards, unpublished observations). The Xho-C fragment appeared to contain three cleavage sites for the purified endonuclease R.CscI, while the vector had none. Two complementary ways of analysis were employed to determine the recognition sequence. For both approaches the Ad5 Xho-C was cleaved with endo R. CscI and the resulting termini were labeled with 32p using T 4 polynucleotide kinase. Singly labeled fragments amenable for sequence determination were obtained by cleavage with endo R.HaeIII. In the first