Regulation of Duodenal Specific Expression of the Human Adenosine Deaminase Gene
1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês
10.1074/jbc.272.42.26634
ISSN1083-351X
AutoresMary R. Dusing, Anthony G. Brickner, Mary Beth Thomas, Dan A. Wiginton,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoFormation 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. 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. 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. A balance of purine and pyrimidine pools in some cells is critical. Excess amounts of some purine compounds cause cell death in developing thymocytes (1Kizaki H. Shimada H. Ohsaka F. Sakurada T. J. Immunol. 1988; 141: 1652-1657PubMed Google Scholar) and in neural cells (2Wakade A.R. Przywara D.A. Palmer K.C. Kulkarni J.S. Wakade T.D. J. Biol. Chem. 1995; 270: 17986-17992Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Insufficient intracellular pools have been shown to be involved in precocious differentiation of cultured intestinal cells (3He Y. Chu S.H. Walker W.A. J. Nutr. 1993; 123: 1017-1027PubMed Google Scholar). In fact, some of the most effective chemotherapy agents are those that are able to interfere with purine metabolism (4Elion G.B. Science. 1989; 244: 41-47Crossref PubMed Scopus (573) Google Scholar). Maintenance of these pools within acceptable limits is accomplished by enzymes of the purine and pyrimidine de novo and salvage pathways. Adenosine deaminase (ADA), 1The abbreviations used are: ADA, adenosine deaminase; bp, base pair(s); kb, kilobase(s); LCR, locus control region; CAT, chloramphenicol acetyltransferase; A/P, anterior/posterior; C/V, crypt/villus. 1The abbreviations used are: ADA, adenosine deaminase; bp, base pair(s); kb, kilobase(s); LCR, locus control region; CAT, chloramphenicol acetyltransferase; A/P, anterior/posterior; C/V, crypt/villus. a member of the purine salvage pathway, catalyzes the irreversible deamination of adenosine and deoxyadenosine. ADA is expressed in all human tissues, yet levels vary in a specific fashion from the highest levels in thymus (790 nmol/min/mg) and duodenum (570 nmol/min/mg) to much lower levels in tissues such as liver (10 nmol/min/mg; Refs.5Adams A. Harkness R.A. Clin. Exp. Immunol. 1976; 26: 647-649PubMed Google Scholar, 6Van der Weyden M.B. Kelley W.N. J. Biol. Chem. 1976; 251: 5448-5456Abstract Full Text PDF PubMed Google Scholar, 7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar). The absence of this enzyme in humans leads to a severe combined immunodeficiency characterized by an absence of both B- and T-cells as well less dramatic changes in other tissues (8Kredich N.M. Hershfield M.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 1. McGraw-Hill, New York1989: 1045-1075Google Scholar). ADA-deficient mice, produced by embryonic stem cell gene-targeting experiments, die at birth with defects of the liver, lungs, and small intestine (9Migchielsen A.A. Breuer M.L. van Roon M.A. te Riele H. Zurcher C. Ossendorp F. Toutain S. Hershfield M.S. Berns A. Valerio D. Nat. Genet. 1995; 10: 279-287Crossref PubMed Scopus (97) Google Scholar, 10Wakamiya M. Blackburn M.R. Jurecic R. McArthur M.J. Geske R.S. Cartwright Jr., J. Mitani K. Vaishnav S. Belmont J.W. Kellems R.E. Finegold M.J. Montgomery C.A. Bradley A. Caskey C.T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3673-3677Crossref PubMed Scopus (123) Google Scholar). Studies in our laboratory have concentrated on the identification and characterization of cis-regulatory elements controlling the tissue-specific pattern of ADA expression. Basal promoter activity for the human ADA gene has been mapped in vitro to 81 bp of 5′-flanking sequence. This basal promoter has no inherent tissue specificity. The proximal promoter region contains no consensus CCAT or TBP binding sites, but it does possess multiple functional Sp1 binding sites that are necessary for both basal and T-cell enhancer-driven activation of this promoter in vitro(11Dusing M.R. Wiginton D.A. Nucleic Acids Res. 1994; 22: 669-677Crossref PubMed Scopus (55) Google Scholar). Neither this minimal promoter nor an additional 3.7 kb of 5′-flanking sequences are capable of consistently activating transcription of a reporter gene in transgenic mice. Even low level activation was absent in a wide variety of tissues examined (7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar). Consistent, high level transgene expression was observed in the thymus of mice with transgenes containing both the promoter fragment and a T-cell-specific cis-regulatory region from the first intron of the human ADA gene (7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar, 12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar). This region directs high level, thymus-specific expression of a linked reporter gene in transgenic mice in an insertion site-independent, copy-proportional manner. It is also able to drive variable low level expression in all tissues examined. Characterization of this region revealed an enhancer element of 230 bp capable of elevating reporter gene expression in transient assay systems and containing consensus binding sites for the E box (13Ephrussi A. Church G.M. Tonegawa S. Gilbert W. Science. 1985; 227: 134-140Crossref PubMed Scopus (491) Google Scholar), Ap1 (14Lee W. Mitchell P. Tjian R. Cell. 1987; 49: 741-752Abstract Full Text PDF PubMed Scopus (1362) Google Scholar), c-Myb (15Howe K.M. Watson R.J. Nucleic Acids Res. 1991; 19: 3913-3919Crossref PubMed Scopus (96) Google Scholar), lymphocyte enhancer-binding factor-1 (LEF-1) (16Giese K. Cox J. Grosschedl R. Cell. 1992; 69: 185-195Abstract Full Text PDF PubMed Scopus (556) Google Scholar), and Ets (17Macleod K. Leprince D. Stehelin D. Trends Biochem. Sci. 1992; 17: 251-256Abstract Full Text PDF PubMed Scopus (289) Google Scholar) families of transcription factors (12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar). Similar factor binding sites are present within the recently characterized mouse homolog to this enhancer (18Brickner A. Gossage D. Dusing M. Wiginton D. Gene (Amst.). 1995; 167: 261-266Crossref PubMed Scopus (14) Google Scholar), and transgenic studies of a segment containing this mouse enhancer demonstrate results analogous to those observed with the human enhancer (19Winston J.H. Hong L. Akroyd S. Hanten G. Waymire K. Overbeek P. Kellems R.E. Adv. Exp. Med. Biol. 1995; 370: 579-584Crossref Scopus (4) Google Scholar). Deletional analysis, footprinting, and site-directed mutagenesis have demonstrated the functional importance of many of these sites within the human enhancer (12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar, 20Ess K.C. Whitaker T.L. Cost G.J. Witte D.P. Hutton J.J. Aronow B.J. Mol. Cell. Biol. 1995; 15: 5707-5715Crossref PubMed Scopus (39) Google Scholar, 21Haynes T.L. Thomas M.B. Dusing M.R. Valerius M.T. Potter S.S. Wiginton D.A. Nucleic Acids Res. 1996; 24: 5034-5044Crossref PubMed Scopus (19) Google Scholar). 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 (12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar,22Aronow B.J. Ebert C.A. Valerius M.T. Potter S.S. Wiginton D.A. Witte D.P. Hutton J.J. Mol. Cell. Biol. 1995; 15: 1123-1135Crossref PubMed Google Scholar, 23Wiginton D. Aronow B. Silbiger R. Potter S. Hutton J. Adv. Exp. Med. Biol. 1991; 309B: 57-60Crossref PubMed Scopus (1) Google Scholar). Compared with the information regarding regulation of human ADA in thymocytes, much less has been discovered about its regulation in other human tissues. A region in the 5′-flank of the mouse ADA gene has been described which is involved in expression in mouse forestomach and fetal placenta (24Winston J.H. Hanten G.R. Overbeek P.A. Kellems R.E. J. Biol. Chem. 1992; 267: 13472-13479Abstract Full Text PDF PubMed Google Scholar, 25Blackburn M.R. Wakamiya M. Caskey C.T. Kellems R.E. J. Biol. Chem. 1995; 270: 23891-23894Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). A placenta-specific regulatory segment was identified recently within that region (26Shi D. Winston J.H. Blackburn M.R. Datta S.K. Hanten G. Kellems R.E. J. Biol. Chem. 1997; 272: 2334-2341Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). ADA is also known to be extremely high in the maternal decidua of mice and other mammals (27Knudsen T.B. Green J.D. Airhart M.J. Higley H.R. Chinsky J.M. Kellems R.E. Biol. Reprod. 1988; 39: 937-951Crossref PubMed Scopus (28) Google Scholar,28Brady T.G. O'Donovan C.I. Comp. Biochem. Physiol. 1965; 14: 101-120Crossref PubMed Scopus (51) Google Scholar) and in human, mouse, and other mammalian small intestine (5Adams A. Harkness R.A. Clin. Exp. Immunol. 1976; 26: 647-649PubMed Google Scholar, 28Brady T.G. O'Donovan C.I. Comp. Biochem. Physiol. 1965; 14: 101-120Crossref PubMed Scopus (51) Google Scholar,29Chinsky J.M. Ramamurthy V. Fanslow W.C. Ingolia D.E. Blackburn M.R. Shaffer K.T. Higley H.R. Trentin J.J. Rudolph F.B. Knudsen T.B. Kellems R.E. Differentiation. 1990; 42: 172-183Crossref PubMed Scopus (55) Google Scholar). Previously, no region of either the mouse or the human ADA genes has been identified which is involved in driving high level expression in either maternal decidua or small intestine. We have now identified and begun characterization of a region of the human ADA gene completely distinct from the T-cell enhancer region which is capable of driving high level, duodenum-specific expression in transgenic mice. p5′acba (12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar) was derived from pADACAT4.0 (7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar) by adding an SalI site at theBamHI site. All subsequent clones are manufactured from this plasmid or one of its derivatives. A 2.3-kbSalI-HindIII fragment (GenBank accession numberM13792, residues 8327–10584; Ref. 30Wiginton D.A. Kaplan D.J. States J.C. Akeson A.L. Perme C.M. Bilyk I.J. Vaughn A.J. Lattier D.L. Hutton J.J. Biochemistry. 1986; 25: 8234-8244Crossref PubMed Scopus (117) Google Scholar) of the human ADA gene containing the human T-cell enhancer and facilitators was isolated from pSph2.3- (11Dusing M.R. Wiginton D.A. Nucleic Acids Res. 1994; 22: 669-677Crossref PubMed Scopus (55) Google Scholar). The ends were filled with dNTPs and Klenow, and BamHI linkers were added. The resulting BamHI fragment was subcloned into the BamHI site of p5′acba to create p5′acba enh. The 13.0-kb SalI insert from λADA4 (GenBank accession number M13792, residues 26353–36741 + 2.6 kb of additional unsequenced 3′-flanking sequences; Ref. 30Wiginton D.A. Kaplan D.J. States J.C. Akeson A.L. Perme C.M. Bilyk I.J. Vaughn A.J. Lattier D.L. Hutton J.J. Biochemistry. 1986; 25: 8234-8244Crossref PubMed Scopus (117) Google Scholar) was cloned into the SalI site of p5′acba enh to create p5′acba enh L4. The 13.0-kbSalI fragment from λADA117 (GenBank accession numberM13792, residues 13518–26509; Ref. 30Wiginton D.A. Kaplan D.J. States J.C. Akeson A.L. Perme C.M. Bilyk I.J. Vaughn A.J. Lattier D.L. Hutton J.J. Biochemistry. 1986; 25: 8234-8244Crossref PubMed Scopus (117) Google Scholar) was subcloned into theSalI site of p5′acba or p5′acba enh to generate p5′acba L117 and p5′acba enh L117, respectively. No positive clones could be detected after repeated transformation into rubidium chloride-treatedEscherichia coli (DH5α, Life Technologies, Inc.). Poor transformation efficiency by this method seemed to be directly related to the large size of these plasmids. Ligation products were introduced successfully into E. coli by electroporation (Bio-Rad), and this method was used routinely. A 7,685-bp HindIII fragment from p5′acba L117 was isolated and ligated to HindIII-cut pUC 18. A 7,685-bp SphI fragment was removed from the resulting plasmid and ligated to SphI-cut p5′acba to create p5′acba L117 Δ1. p5′acba L117 was digested with ApaLI, filled using T4 polymerase and dNTPs, and subsequently digested withSalI to isolate a 8,765-bp fragment. A 49-bpSalI-SphI fragment from pGEM-5Zf- (Promega) was ligated to SalI-SphI-cut p5′acba. The resulting plasmid was digested with EcoRV and SalI and ligated to the 8,765-bp fragment described above to create p5′acba L117Δ2. A 9,612-bp ApaLI fragment from p5′acba L117Δ1 was filled with T4 and dNTPs, digested with SacI, and a 5,723-bp fragment was isolated and ligated into p5′acba plasmid that had been BamHI cut, T4 filled, and then SacI cut to generate p5′acba L117Δ3. All clones were chosen by restriction analysis such that the ADA genomic segments that they contain are in the same relative position and orientation to each other as in the human ADA gene. Plasmid DNAs for transgene isolation were prepared as described previously (12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar). Transgene I has been reported previously as ADACAT4/12 (7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar) and as 3.7/0-IV (12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar). Transgene II also has been published previously as 0.3/II-IIIab (12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar). Both of these transgene fragments were analyzed in C57/C3H hybrid mice. Transgene III is a 17,364-bp BssHII-PvuI fragment from p5′acba enh L4. Transgene IV is a 20,732-bp NdeI-PvuI fragment from p5′acba enh L117. Transgene V is an 18,464-bpNdeI-PvuI fragment from p5′acba L117. Transgene VI is a 14,237-bp NdeI-PvuI fragment from p5′acba L117Δ1. Transgene VII is a 13,149-bp NdeI-PvuI fragment from p5′acba L117Δ2. Transgene VIII is an 8,915-bpNdeI-PvuI fragment from p5′acba L117Δ3. All transgene fragments were isolated from low melting point agarose by digestion with β-agarase, phenol extraction, precipitation, and purification over an Elutip-D column (Schleicher & Schuell). Transgenic mice prepared with Transgenes III–VIII were produced from FVB/N mice. Tail DNA samples from F0 mice bearing Transgenes III or IV were digested with EcoRI, and those containing Transgenes V–VIII were digested with EcoRI and BamHI. DNA was electrophoresed, Southern blotted onto MAGNANT membranes (MSI), and probed with a radiolabeled 1.4-kb EcoRI fragment from pBLCAT6 (31Boshart M. Kluppel M. Schmidt A. Schutz G. Luckow B. Gene (Amst.). 1992; 110: 129-130Crossref PubMed Scopus (230) Google Scholar) encompassing most of the CAT coding sequence. Mice that possessed a band of the appropriate size, 1.9 kb for Transgenes III and IV and 1.4 kb for Transgenes V–VIII, were mated with non-transgenic littermates to establish lines from each founder. Offspring were analyzed by both polymerase chain reaction and Southern blot for transmission of the transgene. F1 transgenic mice were analyzed for CAT and/or ADA activity between 4 and 12 weeks of age except where noted. Small intestine segments were isolated from duodenum (2-cm section adjacent to pyloric sphincter), jejunum (2-cm section from the center of the small intestine), and ileum (2-cm section anterior to the cecum). Other tissues routinely assayed included thymus, liver, tongue, esophagus, colon, and stomach. An extended panel of tissues which was assayed once for each transgene also included quadriceps muscle, lung, heart, ovary/testes, kidney, bone marrow, and brain. For studies examining maternal decidua, transgenic F1 females were superovulated and mated to non-transgenic males. Superovulated but unmated transgenic females were used as control. The uterus was harvested 9.5 days postcoitus and assayed for ADA and CAT. Protein concentrations, CAT activity, and ADA activity were assessed as described previously (7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar). Liver DNA was isolated from the same mice used to determine CAT activity and digested with EcoRI (Transgenes III and IV) or EcoRI andBamHI (Transgenes V–VIII). Samples of 20, 10, 5, 3, and 1 μg 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. Blots were scanned and bands quantitated using a PhosphorImager (Molecular Dynamics, Inc.). A copy number standard curve was generated, and the copy number was determined by averaging the estimated copy number from each lane. Female transgenic mice containing Transgene III, line 1, or Transgene IV, line 2, were superovulated, mated to non-transgenic males, and sacrificed on day 9.5 postcoitus. Superovulated, unmated transgenic females were used as control. 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. Although a 10-fold activation of ADA was observed in pregnant versus non-pregnant uterus, no significant change in CAT reporter gene activity was observed between the two. Low levels of CAT were observed for each, in the range observed for other low level tissues. This low level expression was attributed to the ubiquitous expression generated by the T-cell enhancer/facilitators. Mice from Transgene IV, line 2, were subjected to finer dissection in their analysis. Embryo and yolk sac were dissected from the uterine implantation site. Tissue samples containing the implantation site were dissected away from the non-implantation uterus. Implantation site uterine extracts contained ADA activity (5,240 nmol/min/mg) that was 65 times higher than that from either non-implantation or non-pregnant uterus. Both implantation site and non-implantation site extracts, however, had similar CAT activities that were very low (0.1–0.2% of that in thymic or duodenal extracts). Probes for in situhybridization were prepared from a pGEM-4Z plasmid containing a 550-bpHindIII-NcoI fragment from pSV0-CAT (32Gorman C. Padmanabhan R. Howard B.H. Science. 1983; 221: 551-553Crossref PubMed Scopus (450) Google Scholar) which encompasses the 5′-end of the reporter gene. 35S- or33P-labeled probes were synthesized from linear templates with T7 or SP6 polymerases using an in vitro transcription system (Promega). Duodenal samples from transgenic mice were isolated, rinsed in 1 × phosphate-buffered saline, and fixed in 4% paraformaldehyde in 1 × phosphate-buffered saline at 4 °C overnight, followed by overnight incubation at 4 °C in 30% sucrose, 1 × phosphate-buffered saline. Tissues were frozen in M1 embedding matrix (Lipshaw) and cut into 10-μm sections. Tissue sections were then fixed, acetylated, prehybridized, and hybridized (33Harper M. Masselle L. Gallo R. Wong-Stahl F. Proc. Natl Acad. Sci. U. S. A. 1986; 83: 772-776Crossref PubMed Scopus (535) Google Scholar, 34Hayashi S. Gillam I.C. Delaney A.D. Tener G.M. J. Histochem. Cytochem. 1978; 26: 677-679Crossref PubMed Scopus (218) Google Scholar) with a solution containing 5 × 105 cpm/μl 35S- or 33P-labeled CAT riboprobe. After overnight hybridization at 48 °C, sections were washed at high stringency and treated with RNase A (50 μg/μl, Worthington Biochemical Corp.) and RNase T1 (50 units/ml, Life Technologies, Inc.) at 37 °C for 30 min. Sections were dehydrated and exposed to Kodak NTB-2 emulsion for 1 week at 4 °C. Histological staining of sections was performed with hematoxylin and eosin. Sections were observed by light- and dark-field microscopy. Tissues were isolated from four adult transgenic mice, pooled, and nuclei isolated as described previously (7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar) with the following exceptions. Duodenal mucosa was removed by scraping, and nuclei were isolated from the mucosa only. Polyamine buffer was supplemented with 50 mm N-acetyll-cysteine. PEFABLOC (Boehringer Mannheim) was substituted for phenylmethylsulfonyl fluoride at the same concentration in all solutions. DNase I digestion was carried out on 107 nuclei using 2 units of DNase I in a total volume of 0.4 ml. DNA was digested with XbaI and Southern blotted. A region from the human ADA gene (GenBank accession number M13792, residues 23293–23603; Ref. 30Wiginton D.A. Kaplan D.J. States J.C. Akeson A.L. Perme C.M. Bilyk I.J. Vaughn A.J. Lattier D.L. Hutton J.J. Biochemistry. 1986; 25: 8234-8244Crossref PubMed Scopus (117) Google Scholar) was used as a probe. A 40-kb locus containing the human ADA gene located at 20q13.11 has been cloned and sequenced previously (30Wiginton D.A. Kaplan D.J. States J.C. Akeson A.L. Perme C.M. Bilyk I.J. Vaughn A.J. Lattier D.L. Hutton J.J. Biochemistry. 1986; 25: 8234-8244Crossref PubMed Scopus (117) Google Scholar). A schematic is shown in Fig. 1. A number of fragments from this locus have been included previously in constructions with the CAT reporter gene to identify cis-regulatory elements by testing them in either transient transfection assays or transgenic mouse systems (7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar, 11Dusing M.R. Wiginton D.A. Nucleic Acids Res. 1994; 22: 669-677Crossref PubMed Scopus (55) Google Scholar, 12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar, 22Aronow B.J. Ebert C.A. Valerius M.T. Potter S.S. Wiginton D.A. Witte D.P. Hutton J.J. Mol. Cell. Biol. 1995; 15: 1123-1135Crossref PubMed Google Scholar). A 0.2-kb minimal promoter, defined by transient transfection, is capable of low level reporter gene activation in a number of cell types (7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar). This low level activation is dependent upon the presence of multiple Sp1 binding sites because no TATA binding site is present within this 200-bp fragment (11Dusing M.R. Wiginton D.A. Nucleic Acids Res. 1994; 22: 669-677Crossref PubMed Scopus (55) Google Scholar). The addition of more of the 5′-flanking region up to 3.7 kb did not improve activation levels above this low level observed in transient transfection assays. Neither of these promoter fragments, 0.2 or 3.7 kb, is able to activate even low level reporter gene expression consistently in transgenic mice in a large panel of tissues assayed (7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar). When a 12.0-kb fragment from the ADA first intron (a in Fig. 1) was included 3′ of the CAT reporter in transgenes with either the 3.7-kb (Transgene I, Fig. 1; Ref. 7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar) or the 0.2-kb promoter fragment (12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar), consistent, high level, CAT activity was observed in thymus. Reporter gene expression for these transgenes is both insertion site-independent and copy number-proportional in this tissue (7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar, 12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar). Transgenic mice containing fragment a also expressed the transgene at variable low levels in all non-lymphoid tissues assayed, including duodenum (TableI and Ref. 7Aronow B. Lattier D. Silbiger R. Dusing M. Hutton J. Jones G. Stock J. McNeish J. Potter S. Witte D. Wiginton D. Genes Dev. 1989; 3: 1384-1400Crossref PubMed Scopus (91) Google Scholar). A 2.3-kb fragment (b in Fig. 1) is the smallest subregion of fragmenta which was able to generate a transgene expression profile like that seen with Transgene I (Transgene II in Table I; Ref. 12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar). Further characterization of this 2.3-kb region has revealed a 230-bp core enhancer in the center and facilitators that flank the enhancer core. The facilitator segments have been implicated in assisting in the formation of a region of stable open chromatin at the enhancer and are associated with the LCR-like function that results in position-independent expression of the transgene (12Aronow B.J. Silbiger R.N. Dusing M.R. Stock J.L. Yager K.L. Potter S.S. Hutton J.J. Wiginton D.A. Mol. Cell. Biol. 1992; 12: 4170-4185Crossref PubMed Scopus (93) Google Scholar, 22Aronow B.J. Ebert C.A. Valerius M.T. Potter S.S. Wiginton D.A. Witte D.P. Hutton J.J. Mol. Cell. Biol. 1995; 15: 1123-1135Crossref PubMed Google Scholar). High level repor
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