CARACTERIZACIÓN ESTACIONAL DEL CLIMA EN LA ISLA SANTA

PGEX vectors can be used in bacterial systems to express foreign polypeptides as fusions with glutathione-S-transferase (GST). In general, such fusion proteins are soluble and are easily purified from lysed cells under nondenaturing conditions by absorption with glutathione-sepharose beads, followed by elution in the presence of free glutathione.

3.10.12.1 Induction of expression of GST-fusion proteins

A colony of the pGEX transformant was inoculated into 100 ml LB/ampicillin medium and incubated at 37°C overnight with shaking (200 rpm). The culture was diluted 1:10 into 1 liter of fresh LB / ampicillin medium, incubated at 37°C with shaking for about 1 hr until its OD600

= 0.5. IPTG (100 µM) was given and the culture was incubated at 37°C for an additional 3 hrs. The culture was centrifuged (4500 rpm/10 min, room temperature). The bacterial pellet was washed three times with 50 mM Tris-Hcl, pH8.0 / 100 mM NaCl, resuspended in 10 ml lysis buffer on ice and lysed using a sonicator (15 impulses, 30% output and repeat for 3

times, Branson Sonifier 250). After sonication, the suspension was centrifuged (20000 rpm/30 min/ 4°C, Heraeus HFA 22.50). The GST-fusion protein was purified from supernatant by incubating with glutathione-sepharose beads.

Lysis buffer: 50 mM Hepes, pH 7.9 500 mM NaCl 1 mM EDTA 5 mM DTT 0.5% Nonidet 0.1% Triton X-100

3.10.12.2 Purification of GST-fusion proteins

Glutathione-sepharose beads (Pharmacia) were washed and equilibrated twice with wash buffer. The supernatant from above was incubated with 300-400 µl of equilibrated glutathione-sepharose beads at 4°C for at least 1 hr with gentle shaking. The mixture was centrifuged (2000 rpm/2min/4°C, Heraeus, RS5315). The sepharose pellet was washed with wash buffer for 5 times, it is necessary to incubated the beads with wash buffer for 5 min on ice to destroy the unspecific interactions with the beads. GST-fusion protein was eluted three times by adding the same volume of the elution buffer, mixing well at 4°C for 5 min and centrifuging (2000 rpm/2min/4°C, Heraeus, RS5315). Each Fraction was analyzed by SDS- PAGE. Stored the eluted protein in aliquots containing 10% glycerol at –70°C.

Wash buffer: 50 mM Hepes, pH 7.9 1000 mM NaCl 1 mM EDTA 5 mM DTT 1% Triton X-100 Elution buffer: 150 mM Hepes, pH 7.9 500 mM NaCl 1 mM EDTA 5 mM DTT 1% Nonidet 1% Triton X-100 100 mM glutathione

3.10.12.3 Regeneration of glutathione-sepharose beads

To regenerate glutathione-sepharose beads, the beads were washed with 5 volumes of regeneration buffer and equilibrated with wash buffer.

Regeneration buffer: 50 mM Hepes, pH 7.9 3000 mM NaCl 1 mM EDTA 5 mM DTT 1% Triton X-100

3.10.12.4 Dialysis of GST-fusion protein

Slider-A-Lyzer Dialysis Cassette was used to dialyze GST-fusion protein. Low molecular weight contaminant removal, buffer exchange, desalting and concentration can be accomplished with this device. Dialysis was performed in dialysis buffer at 4°C overnight.

Dialysis buffer: 25 mM Mes, pH 6.5 0.1 mM EDTA 50 mM NaCl 20% glycerol 5 mM MgCl2 1 mM DTT 3.10.13 Unwinding assay

3.10.13.1 Generation of different substrates

3.10.13.1.1 Preparation of 5’-labeled unwinding substrate

The DNA substrate used in unwinding assays consists of 32P labeled complementary oligodeoxynucleotide annealed to single-stranded M13mp19 phage DNA to create a partial duplex. The oligodeoxynucleotide (17-mer) was mainly used except as otherwise stated (Tuteja et al., 1994; Tuteja et al., 1990). A total of 10 pmol of oligonucleotide was labeled at the 5’-end with T4 polynucleotide kinase and [γ32P]-ATP. After 1 hr of incubation at 37°C, the reaction was stopped by heating at 95°C for 20 min. 7.5 µl of M13mp19 DNA and 3.0 µ of annealing buffer were given to the reaction. The mixture was heated at 95°C for 5 min to denature the oligonucleotide and M13mp19. After short centrifugation, it was transferred to a 60°C water bath, slowly cooled and hybridized overnight. Non-hybridized oligonucleotide was removed by gel filtration through a sepharose 4B (Pharmacia) column with elution buffer.

Annealing buffer: Elution buffer:

400 mM Tris, pH 7.5 10 mM Hepes, pH7.4 100 mM MgCl2 100 mM NaCl

1000 mM NaCl 1 mM EDTA

10 mM DTT

3.10.13.1.2 Preparation of blunt ended duplex and small linear DNA unwinding substrate

For the preparation of blunt ended duplex DNA substrate,10 pmol of 17-mer was radioactively labeled and annealed to 40 pmol of complementary 17-mer. Small linear substrates were prepared by labeling 10 pmol of 17-mer and annealing it with 20 pmol of 101- mer.

A total of 10 pmol oligonucleotide (17-mer) was labeled at the 5’-end with T4 polynucleotide kinase and [γ32P]-ATP. After 1 hr of incubation at 37°C, the reaction was stopped by heating at 95°C for 20 min. 20 pmol of oligonucleotide (101-mer) or 40 pmol of oligonucleotide (complementary 17-mer) and 3.0 µl of annealing buffer were given to the reaction. The mixture was heated at 95°C for 5 min to denature the oligonucleotide and M13mp19. After short centrifugtion, it was transferred to a 60°C water bath, which was then turned off. The hybridization was performed overnight.

Non-hybridized oligonucleotide was removed by gel filtration through a sepharose 4B (Pharmacia) column with elution buffer.

3.10.13.2 DNA Unwinding assay

The unwinding assay measures the unwinding of a γ 32P labeled DNA fragment from a partial duplex DNA molecule. The assays were performed in a final volume of 20 µl containing 200 mM Mes (pH 6.5), 10 mM MgCl2, 10 mM DTT, about 200 ng of GST-Purα or truncated

GST-Purα and 32P-labeled unwinding substrate. The reaction was incubated at 37°C for 30 min and terminated by adding 4.0 µl of 5 x loading buffer. The product was separated by electrophoresis on a 12% nondenaturing polyacrylamid gel. A phosphor Image Screen was laid on the gel and followed scanned by Phosphor Image.

5 x loading buffer:

75 mM EDTA 2.25% SDS 37.5% glycerol

3.10.13.3 Generation of point-mutations in a Purαα deletion mutant

Point mutation of Purα cDNA was made using QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene). QuickChangeTM Site-Directed Mutagenesis Kit allows site-specific mutation in virtually any double-stranded plasmid and requires no specialized vectors, unique restriction sites, or multiple transformations.

1) Setting up the PCR reaction:

Two complementary oligonucleotides containing the desired mutation, flanked by unmodified nucleotide sequence were designed. The reaction was prepared as below:

5 µl of 10 x reaction buffer 50 ng of pGPurα

12.5 pmol oligonucleotide primer 1 12.5 pmol oligonucleotide primer 2 0.5 mM dNTP mix

ddH2O to a final volume of 50 µl

1 µl of Turbo Pfu DNA polymerase (2.5 U / µl)

Each reaction was overlaid with 30 µl of mineral oil. 2) Running PCR:

95°C / 1min 1 cycle

95°C / 1min, 65°C / 1min, 68°C / 8 min 16 cycles

68°C / 10min 1 cycle

4°C / ∞

3) Digestion of the products:

1.0 µl of Dpn I restriction enzyme (10 U/µl) was directly added to each amplification reaction below the mineral oil overlay using a small, pointed pipette tip. The mixtures were incubated at 37°C for 1 hr to digest the parental supercoiled dsDNA.

4) After PCR products were transformed into XLI Blue Competent Cells, plasmid DNA was isolated from the cells and sequenced to check the point mutation.

4 Results

SPSF I and II were purified from calf thymus by Dr. Marion Jurk during her Ph.D work in our laboratory. Both proteins bind specifically to ssDNA. Peptide sequences derived from purified SPSF I and II revealed the identity of SPSF I protein with the so-called Hela Purα factor. The DNA binding properties of the Pur protein to single-stranded oligonucleotides of different lengths and sequences were quantified by determination of DNA-binding constants. Hela Purαα cDNA was isolated from a Hela cDNA library and cloned successfully in our laboratory (Jurk et al., 1996).

This dissertation is divided into two parts concentrating on the analysis of Purα’s function. Since Purα has been implicated in diverse cellular functions, including transcriptional activation and repression, translation and cell growth, we decided to generate Purα knock-out (KO) mice to analyze its function in vivo. Thus, the first part of this dissertation reports the generation and characterization of Purα KO mice. While analyzing Purα enzymatic activities, we found accidentally that Purα displayed helicase activity in vitro. Therefore, the second part of this work reports this new property of Purα and the characterization of its function as a DNA unwinding enzyme.

4.1 The generation of Purαα knock-out mice

For the generation of knock-out mice, two essential prerequisites must be fulfilled: (i) The gene to be deleted should be a single copy gene; (ii) the corresponding genomic sequence of this gene locus have to be known. Recent work has shown that mouse Purα is a single copy gene, however, the genomic DNA sequence of Purα is unknown. Thus, this work was started by an analysis of the structure of Purα genomic DNA.