[Frontiers in Bioscience 1, a16-24, January 1, 1996]


Dean C. Ng, Mei-Jhy Su, Richard Kim, and Daniel D. Bikle.

Endocrine Unit, VA Medical Center, University of California, 4150 Clement Street, 111N, San Francisco, CA 94121.

Received 12/7/95; Accepted 12/29/95; On-line 1/1/96


1. Northern Blots
2. Nuclear run-on assays
3. Plasmids and DNA Sequencing.
4. Cell Culture and transfection
5. Reporter assays

Northern blotting

Total RNA was prepared according to the method of Chomczynski and Sacchi (13). Twenty µg total RNA per lane was electrophoresed through 1% agarose-formaldehyde gels. The RNA was then stained with Acridine Orange (Sigma , St Louis, MO) and transferred to Hybond-N+ nylon membranes (Amersham International, Buckinghamshire, UK). The blots were hybridized with the random-primed 32P-labeled cDNA probe PI-2 for involucrin (a gift form Dr. Howard Green, Harvard Medical School, Boston, MA). The amount of involucrin mRNA was quantified by laser densitometry of the resulting autoradiogram.

Nuclear run-on assays.

In vitro transcription studies were performed according to established methods (14,15). Cells were washed with phosphate buffered saline and lysed in a buffer containing 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA and 40% glycerol. The nuclei was then separated by centrifugation and stored in liquid nitrogen until use. Nuclei were thawed on ice and placed in an equal volume of 2X transcription solution (10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 0.3 M KCl, 1 mM ATP, CTP, GTP, and 200 µCi of 32P was then made up to 1 mM CaCl2 and incubated with RNase-free DNase (100 µg/mL) for 5 min. at room temperature. The reaction mixture was then treated with proteinase K (100 µg/mL) in 1% SDS at 37ºC for 30 min., extracted with phenol-chloroform, and ethanol-precipitated. An equal number of counts per min. was hybridized for 48 hrs at 42ºC to the Hybond-N+ (Amersham International, Buckinghamshire, UK) membrane-immobilized pGEM-3Z plasmids containing the involucrin cDNA, cDNA of glyceraldhyde-3-phosphate dehydrogenase (GAPDH), and 18S RNA. pGEM-3Z vector alone was used as a negative control for this assay.

Plasmids Constructs and DNA Sequencing.

We obtained the beta-galactosidase reporter vector that contains 3.7 Kbp of the involucrin gene from Dr. Joseph Carroll, State University of New York, Stony Brook, NY. The 3.7 Kbp fragment of the involucrin gene was sub-cloned from the beta-galactosidase vector (pNassbPL-2) to pGL3-basic to produce a construct with the involucrin promoter in front of a luciferase reporter gene.

pGL3-basic (Promega , Madison, WI) is the parent vector for all the described involucrin constructs in this report. It contains a multiple cloning site in front of the luciferase reporter gene. This vector does not contain any promoter or enhancer and was used as a negative control for all the transfections. The pGL3-control vector is similar to the basic vector except that SV40 enhancer and promoter elements have been incorporated into the vector to increase reporter activity. This vector is used as a positive control for all transfection studies. As a control for transfection efficiency, the vector pRSVbgal, which contains a beta-galactosidase gene that is driven by a Rous Sarcoma Virus promoter and enhancer, was co-transfected with each construct.

Deletion constructs B, C, A-1880, A-979, C-979, A-797, A-156, D, and F were constructed by ligating various restriction fragments of the 3.7 kbp construct (A). Deletion mutations F-2406 and F-2198 were constructed using exonulcease III / Mung bean nuclease treatment of construct F. Oligonucleotides with sequences beginning at -2177, -2131, -2028, -1947 of the involucrin gene were synthesized and used in a polymerase chain reaction (PCR) to generate DNA necessary for the construction of the 5’ deletions F-2177, F-2131, F-2028, F-1947, respectively. Specifically, DNA between -2476 and -1880 of construct F were replaced with the PCR products of these oligonucleotides to generate the respective mutations.

DNA sequencing was done by dye terminator chemistry supplied by Applied Biosystems, Foster City, CA. DyeDeoxy(TM) terminators and AmpliTaq® were used in a sequencing reaction whose products were then loaded onto the Applied Biosystems DNA Sequencer for automated electrophoresis and analysis.

Cell Culture and transfection

Pre-confluent keratinocytes were prepared from cells purified from newborn foreskin as previously described (16). Briefly, foreskins were incubated in 0.25% trypsin at 4ºC overnight to detach the keratinocytes, and the primary cultures were grown to 70-80% confluence in keratinocyte growth medium (KGM, Clonetics, San Diego, CA) containing 0.07 mM CaCl2. Second passage keratinocytes were used in all experiments. The second passage NHK were plated in fresh serum-free KGM with 0.03 mM CaCl2 at a density of 3 X 10(5) cells per 60 mm polystyrene dish (Nunc, Roskilde, Danmark). After 16 hours, the cells were transfected by incubating with fresh medium containing a luciferase reporter plasmid, the pRSVbgal control plasmid, and polybrene (Aldrich, Milwaukee, WI) in a platform rocker at 34ºC for 6 hours (modified from reference (17)). Two µg of each luciferase reporter construct and 0.2 µg of pRSVbgal were co-transfected into each dish of cells. The cells were then exposed to 2 mL of 10% glycerol in KGM for 3 minutes at room temperature and washed twice in 5 mL calcium and magnesium free phosphate buffer saline (PBS) at room temperature before replacing with fresh medium containing 0.03 mM CaCl2 for an additional 16 hours. To determine the effect of calcium on luciferase reporter activity, the medium was either left untreated or supplemented with CaCl2 to a final concentration of 1.2 mM for 24 hrs, and harvested as described below. Cells transfected with up to 4 µg of each plasmid DNA showed a linear increase in their respective reporter activities. High amounts of pRSVbgal DNA (1 µg or more) decreased luciferase reporter activity. When 0.3 µg or less pRSVbgal DNA was used, the inhibitory effect of the pRSVbgal DNA on luciferase activity was not detectable; therefore, a reduced amount of this vector (0.2 µg) was used in all experiments.

Reporter assays

The transiently transfected NHK were harvested 48 hrs after transfection (24 hrs after calcium treatment). Before the harvest, the NHK were washed twice with 5 mL PBS (4ºC) and lysed in 400 µL "reporter lysis buffer" (Promega , Madison, WI) per dish. These cells were then detached from the plate with a cell scraper (Fisher Scientific , Pittsburg, PA), and the lysed cell debris was then removed by centrifugation (2 min. at 14,000 X g). A 10 µL aliquot of each cell extract was used to assay for either beta-galactosidase or luciferase activity using AMPGD (Tropix, Bedford, MA) or luciferin (Promega , Madison, WI), respectively, as substrates. The light output generated by each substrate was quantitated by a luminometer (GEM Biomedical, Inc., Hamden, CT). The relative transcriptional activity of each construct is expressed as the ratio of luciferase activity to beta-galactosidase activity, and normalized to the activity ratio of the 3.7 Kbp construct cultured in 0.03 mM CaCl2.

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