Surjit K. Datta

The use of enzyme therapy to regulate the metabolic and phenotypic consequences of adenosine deaminase deficiency in mice. Differential impact on pulmonary and immunologic abnormalities

Adenosine deaminase (ADA) deficiency results in a combined immunodeficiency brought about by the immunotoxic properties of elevated ADA substrates. Additional non-lymphoid abnormalities are associated with ADA deficiency, however, little is known about how these relate to the metabolic consequences of ADA deficiency. ADA-deficient mice develop a combined immunodeficiency as well as severe pulmonary insufficiency. ADA enzyme therapy was used to examine the relative impact of ADA substrate elevations on these phenotypes. A “low-dose” enzyme therapy protocol prevented the pulmonary phenotype seen in ADA-deficient mice, but did little to improve their immune status. This treatment protocol reduced metabolic disturbances in the circulation and lung, but not in the thymus and spleen. A “high-dose” enzyme therapy protocol resulted in decreased metabolic disturbances in the thymus and spleen and was associated with improvement in immune status. These findings suggest that the pulmonary and immune phenotypes are separable and are related to the severity of metabolic disturbances in these tissues. This model will be useful in examining the efficacy of ADA enzyme therapy and studying the mechanisms underlying the immunodeficiency and pulmonary phenotypes associated with ADA deficiency.

Diverse Genetic Regulatory Motifs Required for Murine Adenosine Deaminase Gene Expression in the Placenta

Murine adenosine deaminase (ADA) is a ubiquitous purine catabolic enzyme whose expression is subject to developmental and tissue-specific regulation. ADA is enriched in trophoblast cells of the chorioallantoic placenta and is essential for embryonic and fetal development. To begin to understand the genetic pathway controlling Ada gene expression in the placenta, we have identified and characterized a 770-base pair fragment located 5.4 kilobase pairs upstream of the Ada transcription initiation site, which directs reporter gene expression to the placenta of transgenic mice. The expression pattern of the reporter gene reflected that of the endogenous Ada gene in the placenta. Sequence analysis revealed potential binding sites for bHLH and GATA transcription factors. DNase I footprinting defined three protein binding regions, one of which was placenta-specific. Mutations in the potential protein binding sites and footprinting regions resulted in loss of placental expression in transgenic mice. These findings indicate that multiple protein binding motifs are necessary for Ada expression in the placenta.

Genetic susceptibility and resistance to diet-induced atherosclerosis and hyperlipoproteinemia.

To test the hypothesis that genetic susceptibility or resistance to diet-induced atherosclerosis is correlated with serum levels of specific lipids, lipoproteins, or apoproteins, male mice of a genetically susceptible and a genetically resistant strain were fed either a normal or an atherogenic diet. After 20 weeks on a normal diet, neither the resistant nor the susceptible strain mice had atherosclerosis; resistant strain mice had serum cholesterol of 66 ± 11 while the susceptible strain mice had 90 ± 1 mg/dl serum cholesterol, and lipoproteins were dominated by a single a-migrating HDL. After 20 weeks on an atherogenic diet, resistant strain mice had 185 ± 55 mg/dl cholesterol, their lipoproteins remained dominated by a-migrating HDL, and two of eight mice had mild atherosclerotic lesions; susceptible strain mice had 510 ± 94 mg/dl cholesterol, multiple a- and pre-R-migrating lipoprotein species, and all 13 had advanced aortic atherosclerosis. The resistant strain mice had an apolipoprotein E/total lipoprotein protein ratio of 0.42 on the normal diet and 0.53 on the atherogenic diet, while the susceptible strain mice had the significantly lower ratios of 0.07 and 0.31, respectively. These data indicate that genetic resistance to diet-induced aortic atherosclerosis in mice is correlated with capacity to prevent large increases in serum cholesterol, to suppress abnormal a- and pre-R-migrating lipoproteins, and to maintain elevated serum apolipoprotein E/total lipoprotein protein ratios. Our data do not preclude the possibility of additional gene control at the level of arterial end organ response.

Impaired Germinal Center Maturation in Adenosine Deaminase Deficiency

Mice deficient in the enzyme adenosine deaminase (ADA) have small lymphoid organs that contain reduced numbers of peripheral lymphocytes, and they are immunodeficient. We investigated B cell deficiency in ADA-deficient mice and found that B cell development in the bone marrow was normal. However, spleens were markedly smaller, their architecture was dramatically altered, and splenic B lymphocytes showed defects in proliferation and activation. ADA-deficient B cells exhibited a higher propensity to undergo B cell receptor-mediated apoptosis than their wild-type counterparts, suggesting that ADA plays a role in the survival of cells during Ag-dependent responses. In keeping with this finding, IgM production by extrafollicular plasmablast cells was higher in ADA-deficient than in wild-type mice, thus indicating that activated B cells accumulate extrafollicularly as a result of a poor or nonexistent germinal center formation. This hypothesis was subsequently confirmed by the profound loss of germinal center architecture. A comparison of levels of the ADA substrates, adenosine and 2′-deoxyadenosine, as well resulting dATP levels and S-adenosylhomocysteine hydrolase inhibition in bone marrow and spleen suggested that dATP accumulation in ADA-deficient spleens may be responsible for impaired B cell development. The altered splenic environment and signaling abnormalities may concurrently contribute to a block in B cell Ag-dependent maturation in ADA-deficient mouse spleens.

An intron 1 regulatory region from the murine adenosine deaminase gene can activate heterologous promoters for ubiquitous expression in transgenic mice

Ubiquitously expressed genes contain regulatory features which allow expression in virtually all cell types. In an effort to understand the molecular basis for this regulatory feature, the chromatin structure of the murine adenosine deaminase gene was examined by DNase I digestion in nuclei of several tissues. The promoter contained a strong hypersensitive site in all tissues examined, including those with very high and very low levels of ADA expression. Transgenic mouse studies revealed that a 3.3 kbEcoRI (3.3 EE) fragment from intron I was required to generate a strong promoter DNase I hypersensitive site, and to produce ubiquitous expression. The 3.3EE fragment also contained a thymic enhancer activity which mapped to sequences conserved with the human ADA gene T-lymphocyte enhancer. Mutational analysis indicated that ubiquitous expression was not dependent on the presence of a functional thymic enhancer. Both the thymic enhancer and the ubiquitous activator within the 3.3EE fragment functioned with heterologous promoters in transgenic mice.

Regulation of Forestomach-specific Expression of the Murine Adenosine Deaminase Gene

The maturation of stratified squamous epithelium of the upper gastrointestinal tract is a highly ordered process of development and differentiation. Information on the molecular basis of this process is, however, limited. Here we report the identification of the first murine forestomach regulatory element using the murine adenosine deaminase (Ada) gene as a model. In the adult mouse, Ada is highly expressed in the terminally differentiated epithelial layer of upper gastrointestinal tract tissues. The data reported here represent the identification and detailed analysis of a 1.1-kilobase (kb) sequence located 3.4-kb upstream of the transcription initiation site of the murineAda gene, which is sufficient to target catreporter gene expression to the forestomach in transgenic mice. This 1.1-kb fragment is capable of directing cat reporter gene expression mainly to the forestomach of transgenic mice, with a level comparable to the endogenous Ada gene. This expression is localized to the appropriate cell types, confers copy number dependence, and shows the same developmental regulation. Mutational analysis revealed the functional importance of multiple transcription factor-binding sites.

COMBINATORIAL INTERACTIONS REGULATE CARDIAC EXPRESSION OF THE MURINE ADENYLOSUCCINATE SYNTHETASE 1 GENE

The mammalian heart begins contracting at the linear tube stage during embryogenesis and continuously pumps, nonstop, throughout the entire lifetime of the animal. Therefore, the cardiac energy metabolizing pathways must be properly established and efficiently functioning. While the biochemistry of these pathways is well defined, limited information regarding the regulation of cardiac metabolic genes is available. Previously, we reported that 1.9 kilobase pairs of murine adenylosuccinate synthetase 1 gene (Adss1) 5′-flanking DNA directs high levels of reporter expression to the adult transgenic heart. In this report, we define the 1.9-kilobase pair fragment as a cardiac-specific enhancer that controls correct spatiotemporal expression of a reporter similar to the endogenousAdss1 gene. A 700-base pair fragment within this region activates a heterologous promoter specifically in adult transgenic hearts. Proteins present in a cardiac nuclear extract interact with potential transcription factor binding sites of this region and thesecis-acting sites play important regulatory roles in the cardiac expression of this reporter. Finally, we report that several different cardiac transcription factors trans-activate the 1.9HSCAT construct through these sites and that combinations result in enhanced reporter expression. Adss1 appears to be one of the first target genes identified for the bHLH factors Hand1 and Hand2.

Development of natural killer cell activity and genetic resistance to bone marrow transplantation with age: effect of neonatal thymectomy.

The effect of neonatal thymectomy on the development of splenic and bone marrow natural cell‐mediated cytotoxicity and on genetic resistance to bone marrow transplantation was examined in mice. Natural cytotoxicity was measured by a 51Cr release assay; the ability to engraft foreign bone marrow was assayed by the spleen colony method. The natural cytolytic response of spleen cells increased progressively from youth to early adulthood, whereas that of the bone marrow declined during the same age period. Neonatal thymectomy significantly elevated the natural killer cell response of young mice only (4 weeks, spleen; 6 weeks, bone marrow). In other experiments, neonatally thymectomized and sham‐operated mice were lethally irradiated at 4 or 6 weeks of age and injected with 2.5, 5.0 or 10 million rat marrow cells. Six days later spleen colonies were markedly reduced in both 4‐ and 6‐week‐old neonatally thymectomized mice with all rat marrow cell doses tested. Neonatal thymectomy did not alter the percentage of erythroid versus other colonies at either 4 or 6 weeks. In both thymectomized and sham‐operated mice the number of colonies increased with increases in marrow cell dose. The data are suggestive of a production and dissemination to the spleen of cells involved in the natural cytotoxic response from the bone marrow.

Adenosine Deaminase-Deficient Mice: Models for the Study of Lymphocyte Development and Adenosine Signaling

PEG-ADA treatment of ADA-deficient mice will serve as a useful in vivo system to biochemically manipulate adenosine levels and signaling. The fact that correction of adenosine levels corresponds with relief of the pulmonary distress, but not immune dysfunction, in ADA-deficient mice suggests that hyperactive adenosine signaling influences the eosinophilia, elevated IgE levels, alveolar defects, and severe inflammation and damage seen in the lungs of the mice. 26

Adenylosuccinate synthetase: A dominant amplifiable genetic marker in mammalian cells

Adenylosuccinate synthetase (AdSS) functions at the branchpoint of purine nucleotide metabolism leading to the synthesis of AMP. The enzyme is inhibited by a metabolite of alanosine, an aspartic acid analog that is highly cytotoxic for most cells. We show here that it is possible to use alanosine selection to isolate from a population of transformants those cells having the highest levels of AdSS activity resulting from uptake and expression of AdSS minigenes. Transformants isolated in this way were selected for resistance to even higher concentrations of alanosine and resulted in the isolation of cells with highly amplified copies of the transfected AdSS minigenes. We demonstrated that nonselectable genes can be cotransferred and coamplified with AdSS minigenes. These findings indicate that AdSS minigenes can be used as dominant amplifiable genetic markers in mammalian cell.