Cloning and Sequence Determination of Catfish Oct1
A cDNA library was prepared in λ ZAPII (Stratagene, La Jolla, CA) from the 42TA catfish monocyte/macrophage line . Approximately 2.5 × 105 pfu were screened at high stringency with a 32P-labeled probe to the POU domain of catfish Oct2 . Two positive clones were obtained and plaque purified. One of these contained a full-length Oct1, and its sequence (GenBank Acc#AJ000267) was determined from both strands by primer-extension sequencing (Biomolecular Resource Laboratory of the Medical University of South Carolina). Isoforms of Oct1 were sought by 3'-RACE using forward primers at positions 1–21 (G-1594); 540–569(G-2338); 547–567 (G-1595); 943–966 (G-2335); 1063–1083 (G-1596); 1836–1856 (G-1597), by 5'-RACE using reverse primers at positions 963–986 (G-2336); 1169–1195 (G-2337) and by RT-PCR using these primers in appropriate combination with reverse downstream primers at positions 615–635 (G-1598); 1206–1226 (G-1599) and 1836–1856 (G-1600). An additional reverse primer in the 3'UTR of the Oct1 clone, G-1593 (5'-AATAAAGTCTAAAGAGCGAGG-3') was also used.
Sequence alignments were carried out using Clustal V in the Megalign suite of programs (DNA Star, Madison, WI) with PAM 250, gap length penalty of 10 and gap penalty of 10. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.1 . The neighbor joining (NJ) phylogenetic tree of Oct1 full-length amino acid sequences was generated with 1000 bootstrap replications. The following amino acid sequences (accession numbers) were used to generate the NJ tree. Catfish Oct2α (Y12651), Catfish Oct2β (Y12652), Catfish Oct1 (AJ00267), Human Oct2 (X13810), Human Oct1 (X13403), Pig Oct2 (Q29013), Pig Oct1 (L38524), Mouse Oct2.1 (X57936), Mouse Oct1 (X68363), Chicken Oct1 (M29972), Xenopus Oct1 (X57165), Zebrafish Oct1 (NM131438), Drosophila Oct2 (S80561), Drosophila Oct1 (S80559), and the Caenorhabditis. elegans homeobox protein (Z79757).
Total RNA was isolated from the head kidney, trunk kidney, spleen, brain, heart, gill and intestine from catfish and from the B lymphoblastoid cell line (1G8) and the T-cell lines (G14D) of the catfish using Trizol (Invitrogen Life Technologies, San Diego, CA). The RNA was then treated with DNase I by addition to RNAeasy mini-kit columns (Qiagen, Valencia, CA). After elution from the columns 0.5 μL of ribonuclease inhibitor, 40u/μl (RNasin, Promega, Madison, WI), was added to the RNA. First strand synthesis was then completed using 4 μL of total RNA, an oligo dT primer, and PowerScript Reverse Transcriptase (BD Biosciences Clontech, Palo Alto, CA). The concentration of cDNA was measured and diluted to 1 μg/μL. A final concentration of 4 ng/μL was used in the PCR reaction with Ex Taq polymerase (Takara, Shiga, Japan). The PCR program was performed for 30 cycles and used the following profile: 30 sec at 95°C; 30 sec at 55°C; 2 min at 72°C. The following primers, located within the C-terminus of the Oct1 gene, were used in the RT-PCR analysis.
G-1392 – 5' CGT CGC TTA CTC CCT CTG CTA C 3'
G-2890 – 5' GAT GCA AGA GCC TGA ATA GTG 3'
G-1480 – 5' CCC ACA CTG TGC CCA TCT ATG A 3'
G-1481 – 5' GAC AGG GAG CCC AGG ATT GAG 3'
Reporter Constructs and Expression Vectors
Three octamer dependent reporter constructs were used. 1) a luciferase expressing construct, pGL3/Δ56/R#2, which contains the core region of the catfish Eμ3' enhancer upstream of the minimal c-fos promoter (described in , 2) a chloramphenicol acetyltransferase expressing reporter, pO3-Δ56-CAT, with a trimer of octamer motifs (ATGCAAAT) upstream of the minimal c-fos promoter , and 3) a chloramphenicol acetyltransferase expressing reporter, pFVH-CAT, with a complete goldfish VH gene promoter . The catfish Oct2β expression vector has been previously described . The catfish Oct1 was directionally cloned (using Not I and Apa I sites) into the pRc/CMV expression vector. Coding sequences were amplified by PCR (forward primer G-2203 – 5' AAG GAA AAA AGC GGC CG C GGC ACC ATG GAG AAT GGA ATA CAT GGA GTC C 3', reverse primer G-2179 – 5' TTT GGG CCC TCA TTG CGC CTT GGA GGC CTC 3'). The restriction sites are underlined and a Kozak consensus sequence is shown in bold in the sense primer. The human BOB.1 vector, expressed from pRc/CMV, was a generous gift from P. Matthias (Friedrich Miescher Institute for Biomedical Research, Novartis Forschungsstiftung, Basel, Switzerland).
For the Gal4 fusion study the reporter construct pG5/CAT was used . This construct contains 5 Gal4 binding sites, located upstream of a minimal TATA box promoter and the CAT reporter gene. The expression constructs for the fusion proteins containing the Gal4 DNA binding domain (DBD) alone or with VP16; nucleolin; catfish Oct2β N-terminus; and catfish Oct2β C-terminus have previously been described . The Gal4DBD-Oct1 domain fusion expression constructs were produced by PCR and were directionally cloned into the Gal4DBD expression construct with XbaI and BamHI restriction enzyme sites. An internal stop codon was also included prior to the BamHI site. All clones were checked by sequencing before use to ensure the absence of PCR-induced mutations.
G – 2977 – 5' TTT TCT AGA GAG AAT GGA ATA CAT GGA GTC CAA 3'
G – 2978 – 5' TTT GGA TCC CTA TAG AGG AAC ATC CAC CCG CTT 3'
Oct1 POU domain
G – 2979 – 5' TTT TCT AGA GTG GAG GAG GCC AGT GAA CTG 3'
G – 2980 – 5' TTT GGA TCC CTA GTT GAT CCT CTT CTC CTT CTG 3'
G – 2981 – 5' TTT TCT AGA AAC CCC CCG AGC AGC AGC ACA 3'
G – 2982 – 5' TTT GGA TCC CTA TCA TTG CGC CTT GGA GGC CAC 3'
Cell Lines and DNA transfection
The catfish B lymphoblastoid cell line 1G8 and T cell line G14D, and the mouse plasmacytoma cell line J558L were maintained as previously described . Transfections were performed with an Electro Cell Manipulator 600 (BTX, San Diego, Ca) and 2-mm gap cuvettes (BTX) were used. All cells were harvested during the logarithmic growth phase and washed once in serum free media (45% AIMV, 45% L15 and 10% deionized water for catfish cells and RPMI-1640 for mouse cells) before resuspension in serum free media prior to transfection. Immediately before transfection 180 μL of cells (8 × 106 cells of 1G8 and 4 × 106 cells of J558L) were mixed with 20 μL of prepared DNA. Reporter construct (2.4 pM of pGL3/Δ56/R#2), (3.2 pM of pFVH-CAT), (3.3 pM of pO3-Δ56-CAT) and empty, Oct1, Oct2 or BOB.1 expression vectors (1.6 pM) were transfected into the cells. For competition studies 1 pM of Oct2 construct was transfected, and increasing amounts of the Oct1 construct were added; 0.5 pM, 1 pM, and 2 pM. For the Gal4 studies 3.95 pM of the pG5/CAT reporter construct was used. For the expression constructs, 1.108 pM of Gal4-DBD alone, Gal4DBD bound to nucleolin, VP16, Oct2β N-Term, Oct2β C-term, Oct1 POU domain, Oct1 N-term, Oct1 C-term was transfected into the catfish 1G8 cell line. In all experiments 1 μg of a Renilla luciferase construct with a CMV promoter (pRL/CMV; Promega) was used as the transfection control. Optimal transfection conditions were used as previously described . Briefly, 1G8 cells were harvested at a density of 3.6–4.0 × 106 cells/ml and were electroporated at 210 V, 1100 μF, and 48Ω. J558L cells were harvested at a density of 0.8–1.0 × 106 cells/ml and electroporated at 130V, 1100 μF, and 48 Ω. All transfected cells were harvested 36–40 hours after electroporation and assessed for expression of the reporter genes.
Luciferase Reporter Assay
Luciferase activity was measured using the Dual-luciferase reporter assay system (Promega) and a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). Transfection activity was normalized to the activity of the co-transfected Renilla luciferase, and the values were calculated as mean ± standard deviation. Statistical analysis was performed using the Student's T-test assuming unequal variances.
Cells were lysed in 1× Reporter Lysis Buffer (Promega) and split into two 100 μl aliquots. One of the aliquots was used in the luciferase reporter assay as described above. The second aliquot was heated at 60°C for 10 min. After heating and centrifugation (16,100 × g for 2 min) the supernatant was transferred into the CAT reaction mixture containing 14C chloramphenicol, n-Butyryl CoA (Promega) and water to a final volume of 125 μL. The reactions were incubated at 37°C for 16 hours and then terminated by the addition of mixed xylenes (Sigma, St. Louis, MO) to extract the modified chloramphenicol. The extracted layer was then added to liquid scintillation fluid and the radioactive product was measured in a liquid scintillation counter. Assay results were normalized using the luciferase assay results and the values were calculated as mean ± standard deviation. Statistical analysis was performed using the Student's T-test assuming unequal variances.
Recombinant protein and antiserum production
The sequence encoding the C-terminal region of catfish Oct1 was digested from the pBluescript vector (Stratagene) as an Eco RV fragment (taking advantage of restriction sites at position 1149–1154 in the Oct1 coding sequence and in the multiple cloning site of the vector) and ligated into the Sma I restriction site of the pQE30 bacterial expression vector (Qiagen) that contains an N-terminal (His)6 tag. The plasmid was transformed into the Escherichia coli M15 strain (Qiagen) and grown at 37°C in LB medium containing a final concentration of 100 μg/ml ampicillin, 25 μg/ml kanamycin, and induced with 2 mM IPTG (Sigma) 5 hours after reaching an OD600 of 0.7. The bacteria were harvested and resuspended in 5 ml sonication buffer (1 mM phenyl methyl sulfonyl fluoride (PMSF), 6 M urea, 400 mM NaCl, 50 mM NaH2PO4 pH 8.0) and lysed by freeze thawing. A 50% TALLON metal affinity resin slurry (BD Biosciences Clontech) equilibrated with sonication buffer was used to purify the His-tagged Oct1 C-terminal peptide. The eluted protein was dialyzed against 50 mM ammonium bicarbonate and subsequently lyophilized. The purified peptide was used to immunize rabbits (Cocalico Biologicals, Reamstown, PA) using primary immunization with complete Freund's adjuvant and boosting using incomplete Freund's adjuvant.
Electrophoretic Mobility Shift Assays
A probe was created to the consensus octamer motif for use in EMSA by annealing the oligonucleotides below (the octamer motif is underlined):
Forward Primer (G-656) – 5' CAATATGAATATGCAAAT TACCT 3'
Reverse Primer (G-567) – 5' CATAGGTAATTTGCAT ATTCATA 3'
A probe with a scrambled octamer sequence was produced by annealing the oligonucleotides below (the scrambled octamer motif is underlined):
Forward Primer (G-1249) – 5' CAATATGAATACAAAATA TACCT 3'
Reverse Primer (G-1250) – 5' CATAGGTATATTTTGT ATTCATA 3'
A probe was created to the first octamer motif located within the core enhancer of Eμ3' using the oligonucleotides below (the octamer motif is underlined):
Forward Primer (G-2110) – 5' GCAAAACACTGCATGTAAAT AGTCTAAT 3'
Reverse Primer (G-2111) – 5' CATTATTAGACTATTTACAT GCAGTGTT 3'
The scrambled probe to the first octamer motif located within the core enhancer of Eμ3' was created using the oligonucleotides below (the scrambled octamer motif is underlined):
Forward Primer (G-2132) – 5' GCAAAACACTGCTATATGAA AGTCTAAT 3'
Reverse Primer (G-2133) – 5' CATTATTAGACTTTCATATA GCAGTGTT 3'
After annealing, the purity of the double-stranded DNA was verified by gel electrophoresis on a 6% non-denaturing polyacrylamide gel (BioRad). The annealed probes were labeled by a radioactive fill-in reaction using the large fragment of DNA polymerase I (Klenow Enzyme, Fisher, Suwanee, GA) and α-32P-dATP (New England Nuclear, Boston, MA) and purified through two Microspin G-25 columns (Amersham Pharmacia Biotech). Nuclear extracts were obtained and EMSA was carried out as described by Ross et al . The TNT quick-coupled transcription/translation system (Promega) was used to generate in vitro synthesized catfish Oct1 and Oct2 proteins. 35S-Methionine (New England Nuclear) labeled in vitro transcribed and translated proteins were analyzed by SDS-PAGE gel electrophoresis and autoradiography. Binding reactions included 4 μl of 5× binding buffer (Promega), 6 μL of TNT products, 2 μL of normal rabbit serum (NRS, anti-Oct1 pre-bleed), cold or scrambled competitor (100 times the concentration of the labeled probe), and anti-Oct1 or anti-Oct2. These reactions were incubated at room temperature for 30 minutes prior to the addition of 1 μL of the radiolabeled probe (106 cpm/μl). After a 30 minute incubation the DNA-protein complexes were analyzed on a 5% non-denaturing polyacrylamide gel, 150V at 4°C with a recirculating cooling system. Gels were washed in 15% methanol, 5% acetic acid for 30 minutes and allowed to dry, before exposure to a Phosphoimager screen and analysis by a Typhoon Variable Mode Imager (Amersham Biosciences) and the ImageQuant software program.