2.1.1 Chemicals
General laboratory chemicals were of analytical grade and purchased from either Merck Ltd., Poole, Dorset, U.K., Boehringer Mannheim, Lewes, East Sussex, U.K., Difco Laboratories, Detroit Laboratories, Detroit, U.S.A. or Sigma Chemical Company Ltd., Poole, Dorset, U.K.. Bovine Serum Albumin was purchased from Advanced Protein Products Ltd.. TEMED was purchased from Sigma Chemical Co. Ltd., Poole, Dorset, U.K.. Hexanucleotides [pd(N)6] for random prime labelling and dNTPs were obtained from Pharmacia Biotechnology Ltd., St. Albans, Herts, U.K.. Radiochemicals, Hybond™"N nylon membranes and Hybond™'C nitrocellulose membranes were obtained from Amersham International Pic., Little Chalfont, Bucks, U.K..
Kodak X-OMAT imaging photographic film was purchased from Sigma Chemical Co. Ltd., Poole, Dorset, U.K. and HyperfilmTM-MP was obtained from Amersham International Pic., Little Chalfont, Bucks, U.K.. Film developing and fixing chemicals were obtained from Photosol, Genetic Research Instrumentation. The Sequenase® version 2.0 sequencing kit was purchased from United States Biochemical Corporation, Cleveland, Ohio, U.S.A.. Protogel and Sequagel concentrate and diluent was purchased from National Diagnostics.
All solid chemicals were dissolved in ddHiO, adjusted to the required pH Avith HCl or NaOH and autoclaved or filter sterilised unless otherwise stated. NaOH and SDS were not autoclaved and phenol, chloroform and ethidium bromide solutions were stored in the dark. Chloroform always contained 4% isoamyl alcohol and phenol contained 0.1% w/v hydroxy quinoline, buffered by shaking three times with an equal volume of 0.5M Tris (pH8.0) and then with 0. IM Tris (pH8.0) removing the aqueous layer each time.
2.1.2 Enzymes
Restriction enzymes and T4 DNA Ligase were obtained from Life Technologies, Paisley, Renfrewshire, U.K. and Promega, Southampton, U.K.. Taq polymerase (for
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PCR) was purchased from United States Biochemical Corporation, Cleveland, Ohio, U.S.A..
DNAase-free RNAase-A (Boehringer Mannheim, Lewes, East Sussex, U.K.) was prepared by dissolving lOmg ml'^ in 15mM NaCl, lOmM Tris-HCl and boiling for 10 minutes to remove and DNAase activity. Aliquots were stored at -20°C.
2.1.3 DNA
All plasmids described below in Table 2.1 were kindly received from the sources indicated.
TABLE 2.1 - DNA Plasmids
PLASMID DESCRIPTION REFERENCE/SOURCE Oct 2.1-bluescript Bluescript KS^ plasmid (Stratagene
Ltd., Cambridge, UK.) containing Oct 2.1 cDNA
Dr. S.J. Dawson,
University College London, London, U.K.
pJ5 Expression vector containing the inducible MMTV promoter with a 3’ polylinker and a transcriptional termination signal.
Morgenstem and Land, (1990)
pJ7 Expression vector containing the constitutive immediate early cytomegalovirus IE94 promoter (CMV) with a 3’ polylinker and a transcriptional termination signal.
Morgenstem and Land, (1990)
Oct2.1-CMV Oct 2.4-CMV Oct 2.5-CMV
pJ7 expression vector into which Oct 2.1, Oct 2.4 and Oct 2.5 cDNA have been cloned.
Dr. K.A Lillycrop,
University College London, London, U.K.
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PLASMID DESCRIPTION REFERENCE/SOURCE pLTR Mammalian expression vector
containing the Moloney murine leukaemia vims promoter, and the reporter CAT gene.
Dr. T.Moroy, Philipps University of Marburg, Germany. Theil et al. (1993) Bm 3a-pLTR Bm 3b-pLTR Bm 3c-pLTR
Full length cDNA sequences of Bm 3a, Bm 3b and Bm 3c cloned into the pLTR vector.
Dr. T.Moroy,
Philipps University of Marburg, Germany. Theil etal. (1993) A-SNAP 6.7 kilobase region of the mouse
SNAP-25 promoter cloned into an expression vector containing the CAT reporter gene.
Dr. M.C. Wilson, The Scripps Research Institute, La Jolla, Cahfomia, U.S.A. Hess etal. (1992)
pBL4.3Syn-CAT 4.3 kilobase region of sy nap sin I gene reporter cloned into an expression vector which contains the CAT reporter gene.
Dr. M.W. Kilimann, Ruhr-Universitat Bochum, Germany.
Hoesche et al. (1995b) pXP2 Expression vector containing the
luciferase reporter constmct.
Dr. AP. Young,
The Ohio State University, Columbus, U.S.A.
X ieetal. (1995) pNOS4.3L 4.3 kilobase region of nNOS
promoter region containing both 5’1 and 5’2 promoters cloned into the pXP2 vector (Figure 6.3).
Dr. AP. Young,
The Ohio State University, Columbus, U.S.A.
Xie etal. (1995) pNOSL 1.842 kilobase region of nNOS 5’2
specific promoter region cloned into the pXP2 vector (Figure 6.3).
Dr. AP. Young,
The Ohio State University, Columbus, U.S.A.
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PLASMID DESCRIPTION REFERENCE/SOURCE pNOSS’lb 300 base pair region of nNOS 5’1
specific promoter region cloned into the pXP2 vector (Figure 6.3).
Dr. A.P. Young,
The Ohio State University, Columbus, U.S.A.
Xie etal. (1995) construct POU The Oct 2 POU domain cloned into
the pJ7 expression vector (Figure 6.6).
Dr. K.A. Lillycrop,
University College London, London, U.K.
Oct2Apl/187-479 The Oct 2 cDNA with the deleted N-terminal region cloned into a mammalian expression vector (Figure 6.6). Dr. P. Matthias, Friedrich Miescher-Institut, Basel, Switzerland. MüUer-Immerglück et al. (1990) Oct2A/2-357 The Oct 2 cDNA with the deleted
C-terminal region cloned into a mammalian expression vector (Figure 6.6). Dr. P. Matthias, Friedrich Miescher-Institut, Basel, Switzerland. Müller-Immerglück et al. (1990) construct A446-463 Oct 2 cDNA with amino acids 446-
463 deleted, cloned into a mammalian expression vector (Figure 6.6).
Prof. R.G. Roeder,
The Rockfeller University, New York, U.S.A.
Gerster et al. (1990) construct A426-463 Oct 2 cDNA with amino acids 426-
463 deleted, cloned into a mammahan expression vector (Figure 6.6).
Prof. R.G. Roeder,
The Rockfeller University, New York, U.S.A.
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PLASMID DESCRIPTION REFERENCE/SOURCE construct A377-463 Oct 2 cDNA with amino acids 377-
463 deleted, cloned into a mammalian expression vector (Figure 6.6).
Prof. R.G. Roeder,
The Rockfeller University, New York, U.S.A.
Gerster et al. (1990)
Table 2.2 shows the sequences of all oligonucloetides used for PCR reactions and sequencing.
TABLE 2.2 - Oligonucleotide sequences
DESCRIPTION OF PRIMERS PRIMER PAIR SEQUENCE 5’^ 3’ SOURCE PCR primers Bm 3a SMART! SMART2 TGCAGAGCAACCTCTTCGCC GAGAT GT GGT CCAGCACCT C Dr. M.D. Smith. Bm 3b 3BNR 3BNF GGTCTGCATCCACGTCGCTC GGAGGGCGAGCTGCTTGAGC Dr. V. Budhram- Mahadeo-Heads. nNOS NOSl N0S2
GCAACAGC GACAAT T T GAG CAGACATAAATGTGGCCTCC Mr. R. Gay. Oct 2 ROM POU GATCAGCAGGATCTCCTCT GGCCCTCAACCTGAGCTTCAAG Dr. K.A. Lillycrop. Ribosomal L6RNA Rib! Rib3 ATCGCTCCTCAAACTTGACC AACTACAACCACCT CAT GCC SNAP-25 SNAPl SNAP2 TGACCAGCTGGCTGATGAGTC CC CAT GT CTAGGGCCATAT GA Dr. N.D. Lakin.
63 DESCRIPTION OF PRIMERS PRIMER PAIR SEQUENCE 5’^ 3’ SOURCE TH THROl THR02 CCTGAGCTTGTCCTTGGC ACATTGGACTTGCATCTCTG Mr. R Gay. Antisense Oct 2 RNA pJ5-PCR pJ5-PCR2
GAT C T GGT AC CAT C GAT GAAT T C AAGCTTCGATCGTCGACTCTAGA
Dr. N.D. Lakin