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Identification of a previously unrecognized production site of human renin
Biochhnica et Biophysica Acta, 1129 (1991) 87-89 © 1991 Elsevier Sciem:c Publishers B.V. All rights reserved 0167-478 i/91/$03.5[)
BBAEXP 90290
87
Rapid Report
Identification of a previously unrecognized production site of human renin Min-Seok Seo i, Akiyoshi Fukamizu ~, Toshikazu Saito 2 and Kazuo Murakami I Institute of Applied Biochen~i.~try, Unicersity of Tsukuba. ibaraki (Japan) and 2 Dicision of Endocrinology and Metabolism, Department of Medicine, Jichi Medical School. Tochigi (Japan) (Received 5 October 1991)
We found expression of the lenin gene in the intestine of human, mouse and the transgenic mouse in which the 3' flanking sequences of the human lenin gene function as a tissue-specific promoter. A cotransfection analysis showed that the promoter is activated by the product of adenovirus EIA 13S mRNA in cells originated from extrarenal tissues. Renin is an;aspartyl p~'oteinase and produced mainly in the kidney, where" synthesis has been localized to the juxtaglomerular (JG) cells [1]. The enzyme is stored and subsequently secreted into the circulation, where it cleaves angiotensinogen, a renin substrate and generates a decapeptide angiotensin I. By angiotensin-converting enzyme, angiotensin 1 is processed to an octapeptide angiotensin !1, which causes arterior vasoconstriction. Thus, the renin-angiotensin system may play an important role in the regulation of blood pressure and possibly contribute to the pathogenesis of hypertension [ 1]. The presence of renin and its mRNA has been demonstrated in a variety of murine extrarenal tissues, such as adrenal, brain, heart, ovary, pituitary, testis and uterus [2-6]. In addition, angiotensinogen mRNA has been also found in the above tissues [7], suggesting that angiotensinogen and angiotensin 11 are locally synthesized. In human, however, the tissue distribution of renin and angiotensinogen mRNAs has not been reported in detail, because of difficulty in obtaining the human samples. Although the exact role of extrarenal renin-angiotensin system is unclear, such system is of particular interest because of the potential importance of its local function. The production of transgenic mice by pronuclear microinjection has provided an especially powerful tool for the study of the regulation of tissue-specific gene expression in mammalian development [8]. In addition, the animal models for human illnesses are useful for studying the pathogenesis of diseases as well as for developing and testing new therapy. As an initial step
Correspondence: K. Murakami, Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
to the production of an animal model for human hypertension, we have recently generated the transgenic mice carrying the human renin gene, with the 3 kb 5' flanking region, whose expression was regulated in a renal-dominant manner [9,10]. Our additional result by a transient transfection analysis showed that the 3 ka upstream sequences fused to the bacterial chloramphenicoi acetyltransferase (CAT) gene could function as a cell type-specific promoter [11,12]. In the present study, we identified an additional production site of human extrarenai renin and tested if an oncogene product, adenovirus EIA 13S mRNA [13], activates the human renin gene promoter in a cultured tumor cell line originated from extrarenal tissues. In the present experiments, total and poly(A) * RNAs were prepared from various mouse tissues, kidney a n d intestine of hRNS-12 mouse [9], and human ileal carcinoma and normal intestines [14] by the guanidinium-cesium chloride method [15]. For examining mouse renin mRNA level, a 284 bp EcoRI-Kpnl fragment was excised from the mouse Renl cDNA [16] was iigated to pGEM4Z (Promega) to construct pGEMmRNI-331. The resulting plasmid was linearized with EcoRI and the uniformly labeled riboprobes were prepared by using T7 RNA polymerase in the presence of [ a - 3 2 p ] C T P (Amersham). To determine expression of the mouse and human renin genes, RNase protection experiments were performed as described previously [9,17]. For CAT assay, pUChRNc:tt30 [12] was cotransfected with pUCl9 (as a control) with or without cAMP (1 raM) or pAdS-13S [13] into HeLa cells by the calcium phosphate precipitation method. Cell extracts were prepared and CAT assay was conducted as described previously [18,19]. The protein concentration was determined using bovine serum albumin as a standard [20].
~8 TABLE ! Tissue distribution o f mouse renin mRNA
510nt P"
Total RNAs (50 p.g) prepared from C57BL/6 were hybridized with Ren-I cRNA probe and analyzed by RNase protection assay. + + + +, very strong expression: + + +, strong expression: + +, moderate expression: + , weak expression: - , not detected.
300nt
Organ
Expression
Heart Intestine Kidney Liver Lung P;incruas Spleen Submandihular ghmd Testis Thymus
+ + + + + +
12
S 4
S
6
78
9
10
Fig. I. Ileal expression of' the human rcnin gene. RNase protcction assay was carried out as described previously [9]. Lanes I to 9, 36 h CXlX~sure: lane I0. 72 h CXlX)surc. Lane I. probe: lane 2, yeast tRNA, 50 p.g: lane 3, transgenic mouse kidney, 50 /~g (T): lane 4, cbXl?4/llincll m~irkcr: l~me 5, nontnmsgcnic mouse kidney, 50 p.g (T): lane 6, transgcnic mouse liver, 50 p,g (T): lane 7, human ileal carcinoma, 50 p.g (T): hmc 8, human intestine, 50 p,g (T): lane 9, human intestine I(H) #g (T): lane I0, transgcnic mouse intestine, I0 p.g (P). T, total RNA: P, poly(A)' RNA.
Transgenic mice have provided a unique opportunity in attempting to identify previously unreported sites of a foreign gene expression in addition to determining the cis-acting sequences of the gene which are required for a regulated expression. In the previous study, we found human renin mRNA in a variety array of the transgenic mouse extrarenal tissues, such as brain, heart, lung, pancreas, spleen, testis and thymus [9]. In the process of further examining an extrarenai expression of the human renin gene in hRN8-12 mouse, we identified mRNA for the transgene in the intestine (Fig. I, lane 10). in the kidney of this mouse, the level of its mRNA was highly detectable (lane 3), whereas the mRNA was undetectable in the transgenic mouse liver (lane 6). Although we have recently reported the ectopic overproduction of human renin by an ileal carcinoma of the patient with the primary reninism [14], we could not detect its mRNA in the normal portion of intestine by Northern blot analysis, probably due to low specificity and sensitivity of this method. Based on the above results obtained from the transgenic mice, we have re-examined expression of the human renin gene in the normal intestine by RNase protection assay. Fig. I in lanes 8 and 9 clearly demonstrated the presence of renin mRNA in human intestine despite much lower level than that of the tumor (lane 7). As listed in Table l, we also found mouse renin mRNA in the intestine by RNase protection assay. These results suggested that the intestine is a previously unrecognized production site of human renin. it has been reported that renin is measured in ovarian renin-secreting tumors [21] and normal ovarian tissues [22]. in the current study, we identified the
+ + + + +
human renin mRNA in both the ileal tumor tissue and its normal portion. Theoretically, tumors arising in any tissues which normally express the renin gene may be able to lead to renin overproduction. These are at least two simple explanations for the overproduction of renin in renin-secreting tumors. First, one cell type that synthesizes renin could grow aberrantly into the transformed cells. In fact, this is the case for the JG cell tumor, a renal renin-secreting tumor, in which we found a large quantity of renin mRNA [23]. Second, some oncogene products in tumor cells might activate the human renin gene expression. To test the second idea, we used an expression vector (pAd5-13S) encoding adenovirus EIA 13S mRNA as a model for oncogene product, because it is implicated in the cellular transformation and the transcriptional activation of some cellular genes and oncogenes during infection [24]. For in vitro transfections, we also used pUChRNcat30 containing the 3 kb 5' flanking region of the human renin gene as a reporter gene [12] and HeLa cells as a model cell line derived from the extrarenal tissues. The cells were cotransfected with the CAT hybrid gene and the oncogene expression vector. As shown in Fig. 2 (lane 2), the promoter activity was not induced by cAMP, although it has been shown that the human renin promoter-CAT fusion gene is activated by cAMP in JEG-3 and placental cells [25,26]. On the other hand, the CAT activity was increased 8-fold in the presence of EIA, suggesting that the renin gene promoter could be activated by oncogene product. However, at present, it is unclear wether adenovirus or some other viruses are involved in the transformation of extrarenai tissues to reninsecreting tumor. While a number ~f studies have demonstrated that many cis-acting sequences of foreign genes act as tissue-specific elements in the transgenic mice [8], an ectopic production of the transgene is frequently ob-
89
1
2
3
We are especiaUy grateful to Prof. Thomas Shenk (Princeton University) for providing us with adenovirus EIA 13S mRNA expression plasmid (pAd5-13S). References
¢ 10 o
llll
0
"0 ,~
5
i
"o i
o IL
% Fig. 2. Eft'cot of adenovirus E IA 13S mRNA product on activation of human renin gene promoter. IteLa cells were transfected with pUChRNcat30 (2.8/ag) in Ihe presence of pUCI t) (0.2 p.g) (hme I ). pUCI9 111.2 p.g) plus I mM cAMP (lane 2) or pAd5-13S ((I.2 p.g) (hme 3) by the calcium phosphate precipitation method. The percent acelylalion of each CAT assay was divided by the basal value (hme I ) to obtain the fi~ld induction. The data shown ;ire represenlalive of fimr independent experiments perfl~rmed in duplicate.
served. For example, anomalous expression of rabbit globin genes has been reported in mouse skeletal muscle and testis where the endogenous globin genes arc not usually expressed, presumably because of an influence of its chromosomal integration sites [27]. Although we cannot rule out the possibility that accumulation of human renin mRNA in the transgenic mouse intestine may result from artifactuallexpression of the transgene, such an explanation for our transgene seems unlikely, because the present result indicates that the endogenous renin mRNA of human and mouse is detected in each intestine. Thus, it would be more likely that a mouse transcription factor may play a regulatory role in the extrarenal expression of the human renin gene in the transgenic mice. In summary, we found expression of the human and mouse renin genes in the intestine through the transgenie technology. The further finding by in vitro transfeet(on study indicated that the oncogene product of adenovirus EIA 13S mRNA activates the human renin gene promoter in cultured tumor cell lir,e. This work was supported by grants from the Ministry of Education, Science and Culture of Japan and Chichibu Cement Co. Ltd. We wish to thank Dr. Kazuhisa Nakayama for generously allowing us use of the mouse renin eDNA for RNase protection assay.
I Reid. i.A.. Morris. B.J. and Ganong, W.F. 11978} Annu. Rev. Physiol. 40. 377-410. 2 Miller. ('.C.J.. Carter. A.T.. Brooks, J.l.. LovelI-Badge. R.II. and Brammar, W.J. (It)St,~) Nucleic Acids Res. 17. 3117-3128. 3 Samani. N.J.. Swales. J.D. anti Brammar. W.J. (1988) Biochem. J. 253. 907-911). 4 Ekker, M.. Tronik. D. Lind Rougeon. F. (Iq89) Proc. Natl. Acad. Sci. USA 86. 5155-5158. 5 Tada, M.. Fukamizu. A.. Seo. M.S.. Takahashi, S. and Murakami. K. (It)St)) Bit~chem. Biophys, Res, ('ommun. 15t,L IlJl~5- l(J71. t~ GIorioso, J.. Atlas. S.N., Laragh, .I.II...le~clewicz. R, and Scalc.~. J.E. (I9/4tr~) Science 233. 1422-1424. 7 ()hkul~o. Ii.. Nakayama. K.. Tanaka. T and Nakanishi, S, (It~St'~) .I. Biol. ('hem. 2t~l. 31~J-323. 8 Palt.ilcr, R.I). and i]rinsler. R.l.. (It)~6) Annu. Rev. (;cnct, 2(I. 41~5-4tit). t) Fukanlilu. A.. Sup. M.S.. ]lalac. T.. Yoko.~am;i. M.. Nonlul;,. I.. Katsuki. M. and Murakami. K. (It)St)) Biochem. l]iophy~. Rcs. ('ommun. Its5. 826-832. I1| Sop. M.S.. Fukamizu. A.. Nomura. T.. Yoko.wlnla. M.. Katsuki. M. Lind Murakami. K. (19911) J. (,ardiovasc. Pharmacol. 16 (Suppl. 4), $8-Si0. II Fukamizu. A.. Ihttae. T., Kon, Y.. Sugimura. M.. Yokoyama. M. Nomura. T.. Kat.suki. M, and Murakami, K. (It)~,~l) Biochem. J. 278. 6111-6113. 12 Fukamizu. A.. Uehara. S.. Sugimura. K.. Kon. Y.. Sugimura. M.. Yokoyama, M., Nomura. T.. Kalsuki. M. and Murakami. K. ( 19t)l ) J. Biol. Regul, I lomeosl. Agents 5. t)7- It)l. 13 ilearing. P. and Shenk. T. (It)85) Mol. ('ell. Biol. 5, 3214-3221, 14 Saito. T,. Fukamizu. A.. Okada. K.. Ishikawa. S.. h~amoh,. Y.. Kuzu~a. T.. Kaxvai. T.. Naruse. K.. Ilirose. S. and Mur;tkanli. K, (itlNg) Endt~cl'il~ol. J;tp. 3(~. 117- 124. 15 ('hirgx~'in..I.M.. PDzybyht. A.E.. MacDonald. R.J. afld Rutlcr. W..I. (lt;7q) I]iocilcnlslr.x IS. 5294 -52qt). It, Kim. W.-S.. Murakami. K. and Nakayama. K. (It)~,q) Nucleic Acids Res. 17. 948t). 17 Krieg. P.A. and Melton. D.A. (It187) Methods Enzymol. 155. 397-415. 18 Gol'mall. ('.. Merlino. G.. Willingham. M.. Pastan. i. and iloward. B. (It182) Mol. Cell. Biol. 2. 11144-11151. I t) Fukamizu. A.. Takahashi. S.. Seo. M.S. Tada. M.. Tan(repro. K.. Uehara. S. Lind Murakami. K. (It)911)J. Biol. Chem. 2(~5. 75707582. 2il Bradlord. M.M. (19761 Anal. Biochem. 72. 248-254. 21 Anderson. P.W.. Macaulay. L.. Do. Y.S.. Shcrrod. A.. D'Ablaing. G.. Koss. M.. Shinagawa. T.. Tran. B.. Montz. F..I. alld I Is(0h. W.A. (19881 Medicine (~8. _57-.68. 22 Do. S.. Sherrod. A.. Lobo. R.A.. Patflson. R.J.. Shinagawa. T.. Chen. S.. Kjt~:;. S. alld llsueh. W.A. (It188) Proc. Natl. Ac;id. Sci. USA 85. 1957-1961. 23 Fukamizu. A.. Nishi. K.. Nishimatsu. S.. Miyazaki. II.. It(rose. S. and Murakami. K. (It)S6) Gene 40. 13t,~- 145. 24 Berk. A.J. (1986) Annu. Rev. Ge,cl. 211. 45-7t). 25 Bur(. D.W.. Nakamura. N.. Kelley. P. and l)zau. V.J. (It)~9).I. Biol. ('hem. 2(~4. 7357-75t~2. 26 Duncan. K.(;.. Ilaidar. M.A.. Baxter..I.!). anti Rctltlclhtd~cr. "1",1~ (It)t)0) Proc. Natl. Acad. Sci. USA 87. 758/4-75t12. 27 Lacy. E.. Roberts. S.. Evans. E.P.. Burtenshaw. M.D. and Constant(n(. F.D. (1t)83) ('511 34. 343-358.
BBAEXP 90290
87
Rapid Report
Identification of a previously unrecognized production site of human renin Min-Seok Seo i, Akiyoshi Fukamizu ~, Toshikazu Saito 2 and Kazuo Murakami I Institute of Applied Biochen~i.~try, Unicersity of Tsukuba. ibaraki (Japan) and 2 Dicision of Endocrinology and Metabolism, Department of Medicine, Jichi Medical School. Tochigi (Japan) (Received 5 October 1991)
We found expression of the lenin gene in the intestine of human, mouse and the transgenic mouse in which the 3' flanking sequences of the human lenin gene function as a tissue-specific promoter. A cotransfection analysis showed that the promoter is activated by the product of adenovirus EIA 13S mRNA in cells originated from extrarenal tissues. Renin is an;aspartyl p~'oteinase and produced mainly in the kidney, where" synthesis has been localized to the juxtaglomerular (JG) cells [1]. The enzyme is stored and subsequently secreted into the circulation, where it cleaves angiotensinogen, a renin substrate and generates a decapeptide angiotensin I. By angiotensin-converting enzyme, angiotensin 1 is processed to an octapeptide angiotensin !1, which causes arterior vasoconstriction. Thus, the renin-angiotensin system may play an important role in the regulation of blood pressure and possibly contribute to the pathogenesis of hypertension [ 1]. The presence of renin and its mRNA has been demonstrated in a variety of murine extrarenal tissues, such as adrenal, brain, heart, ovary, pituitary, testis and uterus [2-6]. In addition, angiotensinogen mRNA has been also found in the above tissues [7], suggesting that angiotensinogen and angiotensin 11 are locally synthesized. In human, however, the tissue distribution of renin and angiotensinogen mRNAs has not been reported in detail, because of difficulty in obtaining the human samples. Although the exact role of extrarenal renin-angiotensin system is unclear, such system is of particular interest because of the potential importance of its local function. The production of transgenic mice by pronuclear microinjection has provided an especially powerful tool for the study of the regulation of tissue-specific gene expression in mammalian development [8]. In addition, the animal models for human illnesses are useful for studying the pathogenesis of diseases as well as for developing and testing new therapy. As an initial step
Correspondence: K. Murakami, Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
to the production of an animal model for human hypertension, we have recently generated the transgenic mice carrying the human renin gene, with the 3 kb 5' flanking region, whose expression was regulated in a renal-dominant manner [9,10]. Our additional result by a transient transfection analysis showed that the 3 ka upstream sequences fused to the bacterial chloramphenicoi acetyltransferase (CAT) gene could function as a cell type-specific promoter [11,12]. In the present study, we identified an additional production site of human extrarenai renin and tested if an oncogene product, adenovirus EIA 13S mRNA [13], activates the human renin gene promoter in a cultured tumor cell line originated from extrarenal tissues. In the present experiments, total and poly(A) * RNAs were prepared from various mouse tissues, kidney a n d intestine of hRNS-12 mouse [9], and human ileal carcinoma and normal intestines [14] by the guanidinium-cesium chloride method [15]. For examining mouse renin mRNA level, a 284 bp EcoRI-Kpnl fragment was excised from the mouse Renl cDNA [16] was iigated to pGEM4Z (Promega) to construct pGEMmRNI-331. The resulting plasmid was linearized with EcoRI and the uniformly labeled riboprobes were prepared by using T7 RNA polymerase in the presence of [ a - 3 2 p ] C T P (Amersham). To determine expression of the mouse and human renin genes, RNase protection experiments were performed as described previously [9,17]. For CAT assay, pUChRNc:tt30 [12] was cotransfected with pUCl9 (as a control) with or without cAMP (1 raM) or pAdS-13S [13] into HeLa cells by the calcium phosphate precipitation method. Cell extracts were prepared and CAT assay was conducted as described previously [18,19]. The protein concentration was determined using bovine serum albumin as a standard [20].
~8 TABLE ! Tissue distribution o f mouse renin mRNA
510nt P"
Total RNAs (50 p.g) prepared from C57BL/6 were hybridized with Ren-I cRNA probe and analyzed by RNase protection assay. + + + +, very strong expression: + + +, strong expression: + +, moderate expression: + , weak expression: - , not detected.
300nt
Organ
Expression
Heart Intestine Kidney Liver Lung P;incruas Spleen Submandihular ghmd Testis Thymus
+ + + + + +
12
S 4
S
6
78
9
10
Fig. I. Ileal expression of' the human rcnin gene. RNase protcction assay was carried out as described previously [9]. Lanes I to 9, 36 h CXlX~sure: lane I0. 72 h CXlX)surc. Lane I. probe: lane 2, yeast tRNA, 50 p.g: lane 3, transgenic mouse kidney, 50 /~g (T): lane 4, cbXl?4/llincll m~irkcr: l~me 5, nontnmsgcnic mouse kidney, 50 p.g (T): lane 6, transgcnic mouse liver, 50 p,g (T): lane 7, human ileal carcinoma, 50 p.g (T): hmc 8, human intestine, 50 p,g (T): lane 9, human intestine I(H) #g (T): lane I0, transgcnic mouse intestine, I0 p.g (P). T, total RNA: P, poly(A)' RNA.
Transgenic mice have provided a unique opportunity in attempting to identify previously unreported sites of a foreign gene expression in addition to determining the cis-acting sequences of the gene which are required for a regulated expression. In the previous study, we found human renin mRNA in a variety array of the transgenic mouse extrarenal tissues, such as brain, heart, lung, pancreas, spleen, testis and thymus [9]. In the process of further examining an extrarenai expression of the human renin gene in hRN8-12 mouse, we identified mRNA for the transgene in the intestine (Fig. I, lane 10). in the kidney of this mouse, the level of its mRNA was highly detectable (lane 3), whereas the mRNA was undetectable in the transgenic mouse liver (lane 6). Although we have recently reported the ectopic overproduction of human renin by an ileal carcinoma of the patient with the primary reninism [14], we could not detect its mRNA in the normal portion of intestine by Northern blot analysis, probably due to low specificity and sensitivity of this method. Based on the above results obtained from the transgenic mice, we have re-examined expression of the human renin gene in the normal intestine by RNase protection assay. Fig. I in lanes 8 and 9 clearly demonstrated the presence of renin mRNA in human intestine despite much lower level than that of the tumor (lane 7). As listed in Table l, we also found mouse renin mRNA in the intestine by RNase protection assay. These results suggested that the intestine is a previously unrecognized production site of human renin. it has been reported that renin is measured in ovarian renin-secreting tumors [21] and normal ovarian tissues [22]. in the current study, we identified the
+ + + + +
human renin mRNA in both the ileal tumor tissue and its normal portion. Theoretically, tumors arising in any tissues which normally express the renin gene may be able to lead to renin overproduction. These are at least two simple explanations for the overproduction of renin in renin-secreting tumors. First, one cell type that synthesizes renin could grow aberrantly into the transformed cells. In fact, this is the case for the JG cell tumor, a renal renin-secreting tumor, in which we found a large quantity of renin mRNA [23]. Second, some oncogene products in tumor cells might activate the human renin gene expression. To test the second idea, we used an expression vector (pAd5-13S) encoding adenovirus EIA 13S mRNA as a model for oncogene product, because it is implicated in the cellular transformation and the transcriptional activation of some cellular genes and oncogenes during infection [24]. For in vitro transfections, we also used pUChRNcat30 containing the 3 kb 5' flanking region of the human renin gene as a reporter gene [12] and HeLa cells as a model cell line derived from the extrarenal tissues. The cells were cotransfected with the CAT hybrid gene and the oncogene expression vector. As shown in Fig. 2 (lane 2), the promoter activity was not induced by cAMP, although it has been shown that the human renin promoter-CAT fusion gene is activated by cAMP in JEG-3 and placental cells [25,26]. On the other hand, the CAT activity was increased 8-fold in the presence of EIA, suggesting that the renin gene promoter could be activated by oncogene product. However, at present, it is unclear wether adenovirus or some other viruses are involved in the transformation of extrarenai tissues to reninsecreting tumor. While a number ~f studies have demonstrated that many cis-acting sequences of foreign genes act as tissue-specific elements in the transgenic mice [8], an ectopic production of the transgene is frequently ob-
89
1
2
3
We are especiaUy grateful to Prof. Thomas Shenk (Princeton University) for providing us with adenovirus EIA 13S mRNA expression plasmid (pAd5-13S). References
¢ 10 o
llll
0
"0 ,~
5
i
"o i
o IL
% Fig. 2. Eft'cot of adenovirus E IA 13S mRNA product on activation of human renin gene promoter. IteLa cells were transfected with pUChRNcat30 (2.8/ag) in Ihe presence of pUCI t) (0.2 p.g) (hme I ). pUCI9 111.2 p.g) plus I mM cAMP (lane 2) or pAd5-13S ((I.2 p.g) (hme 3) by the calcium phosphate precipitation method. The percent acelylalion of each CAT assay was divided by the basal value (hme I ) to obtain the fi~ld induction. The data shown ;ire represenlalive of fimr independent experiments perfl~rmed in duplicate.
served. For example, anomalous expression of rabbit globin genes has been reported in mouse skeletal muscle and testis where the endogenous globin genes arc not usually expressed, presumably because of an influence of its chromosomal integration sites [27]. Although we cannot rule out the possibility that accumulation of human renin mRNA in the transgenic mouse intestine may result from artifactuallexpression of the transgene, such an explanation for our transgene seems unlikely, because the present result indicates that the endogenous renin mRNA of human and mouse is detected in each intestine. Thus, it would be more likely that a mouse transcription factor may play a regulatory role in the extrarenal expression of the human renin gene in the transgenic mice. In summary, we found expression of the human and mouse renin genes in the intestine through the transgenie technology. The further finding by in vitro transfeet(on study indicated that the oncogene product of adenovirus EIA 13S mRNA activates the human renin gene promoter in cultured tumor cell lir,e. This work was supported by grants from the Ministry of Education, Science and Culture of Japan and Chichibu Cement Co. Ltd. We wish to thank Dr. Kazuhisa Nakayama for generously allowing us use of the mouse renin eDNA for RNase protection assay.
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