Abstract
Formation of the mammalian gastrointestinal tract is an ordered process of development and differentiation. Yet, the adult small intestine also retains the plasticity to respond to cues both internal and environmental to modulate intestinal function. The components that regulate this development, differentiation, and modulation at the molecular level are only now being elucidated. We have used the human adenosine deaminase (ADA) gene as a model to identify potential cis-regulatory components involved in these processes within the small intestine. In mammals, high levels of ADA in the small intestine are limited specifically to the differentiated enterocytes within the duodenal region. These studies describe the identification of a region of the human ADA gene, completely distinct from the previously identified T-cell enhancer, which is capable of directing the human intestinal expression pattern in the intestine of transgenic mice. The reporter gene expression pattern observed in these transgenic mice is identical to the endogenous gene along both the cephalocaudal and crypt/villus axis of development. Timing of this transgene activation, however, varies from that of the endogenous mouse gene in that the transgene is activated approximately 2 weeks earlier in development. Even so, this precocious activation is also limited to the epithelium of the developing villi strictly within the duodenal region of the small intestine.
Highlights
Free nucleotides and nucleosides have been shown to be intimately involved in the normal function and regulation of a wide variety of systems, including but not limited to neurotransmission, vasodilation, platelet aggregation, energy transportation, and the synthesis of nucleic acids
Flanking the T-cell enhancer are elements termed facilitators implicated in assisting in the formation of a region of stable open chromatin at the enhancer. These facilitators are associated with an locus control region (LCR) effect that results in position-independent expression of the transgenes containing them [12, 22, 23]
We have identified and begun characterization of a region of the human Adenosine deaminase (ADA) gene completely distinct from the T-cell enhancer region which is capable of driving high level, duodenum-specific expression in transgenic mice
Summary
Plasmid Constructions—p5Јacba [12] was derived from pADACAT4.0 [7] by adding an SalI site at the BamHI site. Liver DNA was isolated from the same mice used to determine CAT activity and digested with EcoRI (Transgenes III and IV) or EcoRI and BamHI (Transgenes V–VIII). Samples of 20, 10, 5, 3, and 1 g of each digested liver DNA and 1, 2, 3, 5, 10, and 25 copy equivalents of a CAT-containing plasmid were electrophoresed, Southern blotted, and probed with the radiolabeled 1.4-kb EcoRI fragment from pBLCAT6. The uterus from a mouse containing Transgene III, line 1, was homogenized in toto and assayed for ADA and CAT and the results compared with the control. In Situ Hybridization—Probes for in situ hybridization were prepared from a pGEM-4Z plasmid containing a 550-bp HindIII-NcoI fragment from pSV0-CAT [32] which encompasses the 5Ј-end of the reporter gene. Human ADA gene (GenBank accession number M13792, residues 23293–23603; Ref. 30) was used as a probe
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