Structural Organization and Chromosomal Assignment of the Swine Endothelin-1 Gene.

Abstract

ABSTRACT. Cosmid clone containing swine endothelin-1 (EDN1) gene, cosEDN1, was isolated from swine cosmid library using swine EDN1 cDNA as a probe. The sequence analysis of cosEDN1 DNA revealed that the swine EDN1 gene consists of 5 exons, spanning approximately 6.5 kb. In the 5'-upstream region of the EDN1 gene, AP-1 and NF-1 elements were found, suggesting the possibility that the expression of swine EDN1 gene is controlled by protooncogene products Fos and Jun, and TGF-β. Fluorescence in situ hybridization (FISH) using cosEDN1 DNA as a probe demonstrated that EDN1 gene resides on swine chromosome 7p13->pter. -KEY WORDS: EDN1, FISH, gene structure, mapping, swine.J. Vet. Med. Sci. 59(6): 431-435, 1997 Mannheim. DNA sequence was determined by dideoxy chain terminator method using PRISM TM ready reaction dyeterminator cycle sequencing kit (Perkin Elmer, U.S.A.) and an ABI 373A DNA sequencer (Perkin Elmer, U.S.A.).Fluorescence in situ hybridization: Peripheral blood cells from boars (Large White) were cultured and labeled with 5-bromodeoxyuridine (BrdU), according to the method described previously [3]. The cultured cells were treated with hypotonic solution and fixative, followed by spreading on glass-slides as described previously [22].The chromosome spreads were treated with 100 µg/ml of RNase A, then with 70% formamide-2 × SSC to denature the chromosomal DNA according to the method of Péquignot [16]. The resulting spreads were subjected to in situ hybridization under the following conditions; 20 µl of hybridization mixture containing 200 ng of heat denatured probe, 40 µg of denatured Cot5 DNA (repetitive sequenceenriched DNA fraction), 2 × SSC, 50 mM sodium phosphate, 1 × Denhaldt's solution, 10% dextran sulfate, 0.1% SDS and 50% formamide was laid on a spread with a cover slip, followed by incubation for 16 hr at 37°C. For the probe, the DNA of the cosmid clone was labeled with biotin-16-dUTP using a nick translation kit (Boehringer Mannheim, FRG). After hybridization, the spreads were rinsed and processed for detection of the hybridization signals on R-banded chromosomes as described previously [3].For precise localization of the signals on chromosomes, the hybridization signals were collected by cooled CCD camera (Hamamatsu Photonics Co., Japan) installed in a microscope under B2 excitation. The same frame of Rband patterns of the chromosome was then collected by the CCD under G-excitation. The image of hybridization signals was superimposed on that of R-band pattern using image analyzer (IBAS; Carl Zeiss, FRG) to locate signal positions on chromosomes.Endothelin-1 (EDN1) consisting of 21 amino acid residues, which is processed from preproendothelin-1 via intermediate protein called "big endothelin-1", was originally identified as a potent vasoconstrictor peptide from a culture supernatant of porcine aortic endothelial cells [20]. Then the EDN1 has been demonstrated to possess a wide variety of biological activities, including contraction of nonvascular smooth muscles, stimulation of proliferation of va...

Notch signaling activation induces cell death in MAPKi‐resistant melanoma cells

“…c‐JUN is a component of AP‐1, a heterodimer consisting of c‐FOS and c‐JUN subunits. Notch has been reported to downregulate AP‐1 complex (Chu & Bresnick, ), and AP‐1 has been shown to regulate EDN1 transcription (Kawana, Lee, Quertermous, & Quertermous, ; Kojima, Ohata, & Yasue, ; Stricklett, Strait, & Kohan, ). Western blot analysis confirmed that NICD1 overexpression caused a marked downregulation in c‐JUN and phospho‐c‐JUN in MAPKi‐resistant MRA‐5 cells, whereas c‐JUN and phospho‐c‐JUN levels showed a slight increase in NICD1‐transduced MAPKi‐sensitive MRA‐6 cells (Figure d).…”

“…To understand the mechanism of action of NICD1 in regulation of EDN1, we identified a list of genes reported to mediate the effect of Notch signaling and also regulate EDN1 based on a curated PubMed search (Poon, Quirk, DeZiel, & Heckerman, 2014). We AP-1 complex (Chu & Bresnick, 2004), and AP-1 has been shown to regulate EDN1 transcription (Kawana, Lee, Quertermous, & Quertermous, 1995;Kojima, Ohata, & Yasue, 1997;Stricklett, Strait, & Kohan, 2011). Western blot analysis confirmed that NICD1 over-…”