Amplification and molecular cloning of murine adenosine deaminase gene sequences

A previously isolated mouse Cl-1D derived cell line (B-1/25) overproduces adenosine deaminase (EC 3.5.4.4) by 3200-fold. The present studies were undertaken to determine the molecular basis of this phenomenon. Rabbit reticulocyte lysate and Xenopus oocyte translation studies indicated that the B-1/25 cells also overproduced adenosine deaminase mRNA. Total poly(A+) RNA derived from B-1/25 was used to construct a cDNA library. After prehybridization with excess parental Cl-1D RNA to selectively prehybridize nonamplified sequences, 32P-labeled cDNA probe synthesized from B-1/25 total poly(A+) RNA was used to identify recombinant colonies containing amplified mRNA sequences. Positive clones containing adenosine deaminase gene sequences were identified by blot hybridization analysis and hybridization-selected translation in both rabbit reticulocyte lysate and Xenopus oocyte translation systems. Adenosine deaminase cDNA clones hybridized with three poly(A+) RNA species of 1.5, 1.7, and 5.2 kilobases in length, all of which were overproduced in the B-1/25 cell line. Dot blot hybridization analysis using an adenosine deaminase cDNA clone showed that the elevated adenosine deaminase level in the B-1/25 cells was fully accounted for by an increase in adenosine deaminase gene copy number. The adenosine deaminase cDNA probes and the cell lines with amplified adenosine deaminase genes should prove extremely useful in studying the structure and regulation of the adenosine deaminase gene.     

Preliminary X-ray analysis of crystals of murine adenosine deaminase

We have obtained single crystals of a cloned mammalian adenosine deaminase (Mr = 41,000), a key enzyme in purine degradation and in normal development of the immune system, that are suitable for high-resolution structural analysis. The crystals belong to the space group C2 with unit cell parameters a = 101.68 A (1 A = 0.1 nm), b = 94.38 A, c = 85.51 A, and beta = 96.54 degrees. The asymmetric unit contains two enzyme molecules.     

Fine mapping and functional activity of the adenosine deaminase origin in murine embryonic fibroblasts

DNA replication initiates at origins within the genome. The late-firing murine adenosine deaminase (mAdA) origin is located within a 2 kb fragment of DNA, making it difficult to examine by realtime technology. In this study, fine mapping of the mAdA region by measuring the abundance of nascent strand DNA identified two origins, mAdA-1 and mAdA-C, located 397 bp apart from each other. Both origins conferred autonomous replication to plasmids transfected in murine embryonic fibroblasts (MEFs), and exhibited similar activities in vivo and in vitro. Furthermore, both were able to recruit the DNA replication initiator proteins Cdc6 and Ku in vitro, similar to other bona fide replication origins. When tested in a murine Ku80(-/-) cell line, both origins exhibited replication activities comparable to those observed in wildtype cells, as did the hypoxanthine-guanine phosphoribosyltransferase (HPRT) and c-myc origins. This contrasts with previously published studies using Ku80-deficient human cells lines and suggests differences in the mechanism of initiation of DNA replication between the murine and human systems.     

Sequence, genomic organization and functional expression of the murine tRNA-specific adenosine deaminase ADAT1

We have recently identified the first mammalian tRNA-specific adenosine deaminase human ADAT1, a member of the ADAR family of RNA editing enzymes. This protein is responsible for the first step of the unique A(37) to m(1)I(37) modification in eukaryotic tRNA(Ala). Here, we present the genomic structure of murine ADAT1 and the functional expression of mADAT1 cDNA. In mouse, as well as in human, ADAT1 is expressed from a single copy gene. The coding region of the mADAT1 gene is spread over nine exons, covering approximately 30kb of genomic DNA and encodes a protein of 499 amino acids. Overall, mADAT1 shares 81% nucleotide homology and 87.5% protein homology with the human ortholog. The recombinant mouse protein is active specifically and with a high efficiency on human tRNA(Ala) in vitro. Its genomic organization is compared to the structures of the sequence-related, pre-mRNA specific adenosine deaminases ADAR1 and ADAR2.     

Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli

Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%.     

Function of murine adenosine deaminase in the gastrointestinal tract

Adenosine deaminase (ADA) deficiency in humans leads to a combined immunodeficiency characterized by severe T and B cell lymphopenia. ADA-deficient humans also display defective development of gut-associated lymphoid tissues (GALT). They lack lymphoid cells, and the Peyer's patches are without germinal centers. In mice, ADA-deficient fetuses die perinatally due to liver damage, but they also exhibit pathology in the thymus, spleen, and the small intestine. The GI phenotype associated with ADA-deficient humans prompted us to examine the effect of ADA-deficiency on mouse small intestine tissue. The work presented here focuses on understanding the physiological role of ADA in the GI tract, using ADA-deficient mice rescued from perinatal lethality by restoring Ada expression to trophoblast cells. Histologically and immunologically, the GALT was compromised at all sites in ADA-/- mice, with the most dramatic changes seen in the Peyer's patches. Profound disturbances in purine metabolism were detected in all the gastrointestinal tissues. In particular, adenosine and deoxyadenosine, the ADA substrates, increased markedly while the product inosine decreased. The activity of S-adenosylhomocysteine hydrolase decreased throughout the GI tract, indicating a possible disruption of cellular transmethylation and activation of apoptotic pathways. There were also disturbances in the purine metabolic pathway with a decrease in the production of downstream nucleosides hypoxanthine and xanthine.     

Different functional subsets of cultured murine T cells express characteristic levels of adenosine deaminase activity

The level of adenosine deaminase (ADA) activity in mouse T-lymphocyte cultures was studied under different growth-supporting conditions and in mixed lymphocyte culture-derived long-term T-cell lines and clones. Early after the initiation of in vitro culture, the levels of ADA (2000 U/mg) were similar in bulk cultures either depleted or not depleted in Lyt-2+ T cells. Enrichment for cytolytic T lymphocytes (CTL) obtained by addition of exogenous interleukin 2 (IL-2), was accompanied by a net decrease of ADA activity (110 +/- 15 U/mg). All the tested CTL-A lines derived from such cultures were also characterized by a low or undetectable level of this enzyme (at best 160 +/- 70 U/mg) as previously observed. In contrast, "Lyt-2-" T-cell bulk cultures grown, without addition of exogenous IL-2, in the presence of gamma-irradiated H-2d stimulators maintained a constant level of ADA activity (1770 +/- 340 U/mg) for at least 3 months. Functionally distinct types of Lyt-2- T-cell lines were also analyzed: T-cell lines competent to activate B lymphocytes to growth and terminal maturation as well as others devoid of detectable functions showed a stable ADA level comparable to that expressed by the original bulk culture 1685 +/- 620 U/mg). The present results demonstrate that, like tumor cell lines, most normal T lymphocytes express a high level of ADA activity in culture, which strongly suggests that the low level of ADA activity exhibited by CTL is a characteristic of this functional subset.     

Structural basis of compound recognition by adenosine deaminase

Structural snapshots corresponding to various states enable elucidation of the molecular recognition mechanism of enzymes. Adenosine deaminase has two distinct conformations, an open form and a closed form, although it has so far been unclear what factors influence adaptation of the alternative conformations. Herein, we have determined the first nonligated structure as an initial state, which was the open form, and have thereby rationally deduced the molecular recognition mechanism. Inspection of the active site in the nonligated and ligated states indicated that occupancy at one of the water-binding positions in the nonligated state was highly significant in determining alternate conformations. When this position is empty, subsequent movement of Phe65 toward the space induces the closed form. On the other hand, while occupied, the overall conformation remains in the open form. This structural understanding should greatly assist structure-oriented drug design and enable control of the enzymatic activity.     

Characterization and functional analysis of the murine Frat2 gene

The Frat1 proto-oncogene was first identified as a gene contributing to tumor progression in T-cell lymphomas induced by retroviral insertional mutagenesis with the Moloney murine leukemia virus. The biological function of Frat remained elusive until its Xenopus homologue GBP was isolated as a glycogen synthase kinase 3 (GSK3)-binding protein and was shown to be an essential component of the maternal Wnt-signaling pathway. To date two Frat homologues have been described in the mouse, Frat1 and Frat3. The proteins encoded by these two genes are 84% identical. Here we describe the cloning and characterization of a third murine Frat homologue, Frat2, which is the mouse ortholog of human FRAT2. Frat1 and Frat2 are juxtaposed on chromosome 19 in a chromosomal organization conserved between man and mouse. We show that Frat1 and Frat2 are phosphorylated, which is the first evidence that these proteins are subject to posttranslational modification. Like Frat1, Frat2 is able to bind to GSK3beta. However, a side-by-side comparison of the murine Frat proteins for their capacity to induce signaling through beta-catenin/T-cell factor reveals that Frat2 is a less potent activator of the canonical Wnt pathway. Frat2 protein accumulates to higher levels upon transfection into 293T cells than either Frat1 or Frat3. Thus, whereas Frat1 may be a core component of canonical Wnt-signaling, Frat2 might very well be part of a divergent intracellular GSK3beta pathway.     

Structural and conformational analysis of pentostatin (2'-deoxycoformycin), a potent inhibitor of adenosine deaminase

X-ray, NMR and molecular mechanics studies on pentostatin (C11H16N4O4), a potent inhibitor of the enzyme adenosine deaminase, have been carried out to study the structure and conformation. The crystals belong to the monoclinic space group P21 with the cell dimensions of a = 4.960(1), b = 10.746(3), c = 11.279(4)A, beta = 101.18(2) degrees and Z = 2. The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.047 for 997 reflections. The trihydrodiazepine ring is nonplanar and adopts a distorted sofa conformation with C(7) deviated from the mean plane by 0.66A. The deoxyribose ring adopts a C3'-endo conformation, different from coformycin where the sugar has a C2'-endo conformation. The observed glycosidic torsion angle (chi = -119.5 degrees) is in the anti range. The conformation about the C(4')-C(5') bond is gauche+. The conformation of the molecule is compared with that of coformycin and 2-azacoformycin. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. The conformation of deoxyribose in solution is approximately 70% S and 30% N. Molecular mechanics studies were performed to obtain the energy minimized conformation, which is compared with X-ray and NMR results.