CONSTRUCTS

2.4a Cloning o f the 5' region o f 5a-reductase I gene into pGL2Basic reporter plasmid

To study the promoter activities of the genomic DNA fragments isolated from 70

screening the library, DNA sequences that were upstream of the ATG start site needed to be cloned into a reporter plasmid. The putative promoter regions were cloned into the promoterless pGL2Basic plasmid containing the luciferase reporter gene (see map in previous section). The multiple cloning site (MCS) is present before the luciferase gene in the plasmid; therefore the effectiveness of a cloned promoter at transcribing the luciferase gene can be measured.

To clone only the sequence 5'of the ATG site of the 9kb Not I genomic DNA fragment cloned into pBS, a PCR and restriction digestion based strategy was used. A 5a-reductase I specific 3' primer complementary to bases -22 to -1 next to a Bgl H restriction site was synthesised (described earlier). Since a Bgl H restriction site is present on the 3' end of the MCS of pGL2Basic (see map), it can be utilised to clone the 3' end of the fragment into this vector.

-22 -1 B g /n

I---1---1 5a-reductase I specific 3' primer.

Using the universal primer (M l3-20 primer complementary to sequences at the start of multiple cloning site on pBS) as the 5' primer and the 9kb Not I fragment cloned into pBS as a template, a PCR reaction was carried out. l(X)|xl of PCR reaction mixture contained 200ng of template DNA, lOpmol of each primer, KXlpM dNTP, 10)il of lOX PCR buffer and 5U of Taq polymerase. PCR was carried out at 94°C for 1 minute, 63°C for 1 minute, 72°C for 1 minute for 30 cycles followed by a final extension step of 72°C for 10 minutes. lOjil of the reaction mixture was checked on a 4.5% polyacrylamide gel and a ~0.75kb PCR product seen. Then the remaining products were run on the gel, the band excised and processed as described in isolation and reamplification of PCR products from gels (described above).

To clone the 5' end of the fragment into pGL2Basic, a restriction digestion strategy was used. 5' of the N ot I site in the MCS of pBS (used to clone the genomic fragment into this plasmid), there is a Xba I site (See map of pBS). Although a site for Xba I is not present on pGL2Basic, there is a Nhe I site 5' of the Bgl II site which produces ends compatible with

Xba I. Therefore 3|Xg of pGL2Basic was digested B gl II and Nhe I in a 20|xl reaction mixture containing 5U of each enzyme and 2|l i1of appropriate lOX buffer at 37°C for 2 hours. The cleaned 0.75kb PCR product was also digested with 5U each of Bgl II and Xba I and l|il of each digest checked on a 1% agarose gel. Both the digested pGL2Basic and the PCR product were then extracted with an equal volume of phenol:chloroform:IAA (25:24:1), vortexed and centrifuged at 13,000 rpm for 10 minutes to remove excess enzymes. The top layer was then extracted once with an equal volume of chloroform:lAA (24:1) to remove the phenol and centrifuged at 13,000 rpm for 5 minutes. The DNA was precipitated with 1/10 volume of 3M sodium acetate, pH 5.2 and 2 volumes of 100% ethanol at room temperature (RT) for 20 minutes and centrifuged for 30 minutes to pellet the DNA. The supernatant was carefully removed with a pipette and the pellets air dried for 5 minutes. The pellets were redissolved in lOpl of water. A ligation reaction was carried out as described using 3 times excess of PCR product compared to pGL2Basic. The ligated product was used to transform competent E. coli cells, isolated colonies grown and miniprep DNA extracted. The resulting plasmid DNA was checked by digesting with restriction enzyme. It was also prepared for sequencing using the original PCR primers. The sequencing reaction showed that only 587bp of the original 9kb Not I fragment that hybridised to the probe was 5' to the ATG site and the resulting plasmid was called pGL5ocRI0.59.

Cloning o f the 4.6kb 5 a-reductase I promoter construct

Since the 4kb N ot I fragment is immediately upstream of the 587bp fragment already cloned into pGL5ocRI 0.59, it was cloned upstream of the smaller 587bp promoter fragment to create a larger 5a-reductase I promoter construct. 3|Xg of pGL5(xRI 0.59 was cut with

Not I and dephosphorylated. 20|Xg of phage DNA from P2 was digested with Not I and separated on an agarose gel. The band corresponding to the 4kb fragment was excised and the DNA gel extracted using the QIAEX II kit. Ligation reactions were set up using the 4kb fragment with the Not I cut and dephosphorylated pGL5ocRI 0.59 and used to transform competent E. coli cells. Resulting colonies were picked, minipreped and the miniprep DNA digested with enzymes to check insert sizes. Clones with the correct sized fragments were sequenced and found to contain the 4.59kb 5a-reductase I promoter sequence in the correct orientation. This vector was called pGL5(xRI 4.6.

2.4b Cloning o f the 5' region of 5a-reductase II gene into pGL2Basic reporter plasmid

To clone the sequence 5'of the ATG site of the 5kb Sac I genomic DNA fragment in pBS, a PCR and restriction digestion based strategy was used, as for the type I promoter. A 5a-R II specific 3' primer complementary to bases -25 to -1 next to a Bgl II restriction site was synthesised.

-25 -1 BglU

I- - - 1- - - 1

5a-reductase II specific 3' primer.

Using the universal primer as the 5' primer and the 5.5kb Sac I fragment cloned into pBS as a template, a PCR reaction was carried out. lOOfil of PCR reaction mixture contained 200ng of template DNA, lOpmol of each primer, lGG|iM dNTP, 10|xl of lOX PCR buffer and 5U of Taq polymerase. PCR was carried out at 94°C for 1 minute, 62°C for 1 minute, 72°C for 1 minute for 30 cycles followed by a final extension step of 72°C for 10 minutes. lOjil of the reaction mixture was checked on a 4.5% polyacrylamide gel and a -O.Skb PCR product seen. The remaining products were then run on the gel, the band excised and processed as described in isolation and reamplification of PCR products from gels. Since pGL2Basic contains a Sac I site, both this plasmid and the PCR product were digested with

Sac I and Bgl n, the products purified and ligated. Ligated product was used for transformation and miniprep DNA of isolated colonies sequenced. Only 743bp of the 5.5kb

Sac I fragment that hybridised to the probe was 5' to the ATG site and the resulting plasmid was called pGL5otRII0.75.

2.4c Generation o f smaller 5a-reductase I and II deletion constructs

Once the 4.6kb and 0.75kb pGL2Basic constructs of the 5a-reductase I and II promoters were made, smaller constructs were prepared utilising restriction enzyme sites present in both the pGL2Basic plasmid and the promoter regions. The procedure for one such construct, pGL5oRI 0.3 is described below. In the pGL2Basic MCS, there is a Sac I site upstream of the Bgl II site. The sequence of the 0.59kb construct showed that there is a Sac

I site 302 bp upstream from the ATG site. pGL5otRI 0.59 vector was digested with Sac I which cut at both the promoter and at the MCS to excise the 285bp distal promoter fragment, leaving the first 302bp of 5a-Reductase I promoter in the pGL2Basic vector. 3p,g of pGL5ocRJ 0.59 was digested in a 20|Xl reaction volume with 5U of Sac I and 2|il of lOX buffer at 37°C for 2 hours. Ijxl of the digested product was checked on a 0.8% agarose gel. The remaining digest was also electrophoresed and the corresponding band was excised and gel cleaned using the QIAEX II kit. To 1|li1 of the gel cleaned plasmid Ipl of T4 DNA ligase and 2pl of 10 X T4 DNA ligase buffer were added and made up to lOpl with water and incubated at 15°C overnight for blunt end ligations or 22°C for 3 hours for sticky end ligations. Ipl of the ligation mix was used to transform competent E. coli cells, colonies were picked and miniprep DNA digested with Sac I and other enzymes to check insert size. Colonies containing the correct sized insert of 0.3 kb were sequenced using pGL2Basic sequencing primers. This vector was called pGL5oRI 0.3.

2.5 TRANSFECTION AND ASSAY OF PUTATIVE 5a-REDUCTASE

PROMOTER CONSTRUCTS IN CELL LINES