EP 0 314161 A1
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`this vector. Both of these goals could be accomplished by the removal of a 121 bp Hind ill-Bgl ll fragment
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`located at the 5’ end of the Ecogpt gene. This 121 bp fragment contains two initiation codons upstream of
`the functional
`initiation codon (which is not on the 121 bp fragment). Removal of these upstream sites
`should increase levels of expression of the Ecogpt gene. Briefly, this was accomplished by digesting pSV2-
`gpt with Hind ill and Bgl
`ll, making the termini blunt ended with the Klenow fragment of DNA polymerase,
`self-ligating the DNA and transforming competent bacteria. The experimental details follow.
`Ten micrograms of pSV2-gpt DNA (ATCC No. 37145) were digested to completion with Hind ill (80
`units for 1 hour at 373C). The Hind Ill-digested material was then digested to completion with Bgl
`ll (24
`units for 30 minutes followed by 16 units for 1 hour at 37“ C). The digested material was fractionated on a
`1% agarose gel in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA). After staining with ethidium bromide at
`0.25 ug/ml, DNA fragments of 5.4 kb and 121 bp were visualized. The higher molecular weight band was
`electrophoresed into NA45 DEAE paper (Schleicher and Schueli) and then eluted from the paper according
`to the manufacturer's instructions. The DNA was precipitated with ethanol and resuspended to approxi-
`mately 1 ug/ui in TE buffer (10 mM Tris, pH 7.8. 1 mM EDTA).
`The termini created by the restriction endonucleases were made to be blunt ended using the Klenow
`fragment of DNA polymerase (2 units enzyme per 1 ug of DNA in 25 LLI buffer (50 mM Tris, pH 7.2, 10mM
`M9804, 0.08 mM dATP, 0.08 mM dCTP, 0.08 mM dTTP, 0.08 mM dGTP, 0.1 mM DTT, 50 ug/ml BSA) for
`10 minutes at room temperature). The sample was immediately phenol and chloroform extracted and then
`precipitated with ethanol. The DNA pellet was resuspended in TE to 1 ug/LLI. One microgram of DNA was
`self-ligated in a final volume of 20 LLI at 15° C for 7 hours using 1 unit T4 DNA ligase (Bethesda Research
`Laboratories) in "ligation buffer" (50 mM Tris, pH 7.8, 10 mM M902, 1 mM DTT, 6 mM KCl, 1 mM ATP, 1
`mM spermidine, and 5% polyethylene glycol (Sigma PEG 8000)).
`The ligation reaction was diluted 1: 5 in Te and 1 lJ.l was used to transform 100 ul of competent E. co__i_i
`DH5a cells (Bethesda Research Labs) according to manufacturer'5 instructions. Transformed bacteria were
`plated on LB agar containing 100 ILg/mi ampicillin. Plasmid DNA from ampicillin resistant colonies was
`screened for its ability to be cut with EcoR l, but not with Hind Ill or Bgl. ii. The resultant vector was termed
`pG.
`
`“.292 p.51.CG. (jets)
`
`The pBR322 sequences of pG were replaced with the analogous sequences from pUC18 to increase
`yields of plasmid DNA. Briefly, this was accomplished by digesting pG with EcoR I and Pvu ll, recovering
`the 3 kb DNA fragment containing the Ecogpt gene and SV40-derived DNA, and ligating it to the Pvu lI
`fragment from pUC18 containing the origin of replication and ampicillin resistance gene.
`The details were as follows: Ten micrograms of pUC18 (Bethesda Research Laboratories) were
`digested to completion with Pvu ll (50 units for 2 hours at 37°C) and 10 ug of pG were digested to
`completion with EcoR | (50 units for 2 hours at 37° C), and then with Pvu ll (50 units for 2 hours at 37° C).
`Both digests were electrophoresed through 1.2% agarose and the desired fragments (3 kb for p6 and 2.4
`kb for pUC18) were electrophoresed into a NA45 DEAE membrane. The DNA was then eluted from the
`DEAE paper according to the manufacturer's instructions, ethanol precipitated and resuspended to 1 ug/ul
`in TE buffer.
`
`The EcoR 1 ends from the digested pG were filled in using the Klenow fragment of DNA polymerase (2
`units of enzyme and 1 ug DNA for 10 minutes at room temperature in a 25 Lil reaction volume). The
`sample was then phenoi and chloroform extracted, ethanol precipitated and resuspended to 200 ng/ui in TE
`buffer. Two micrograms of the pUC18 fragment were treated with calf intestinal alkaline phosphatase (CIP;
`Boehringer-Mannheim) suspended in 50 mM Tris-Cl, pH 9.5,
`1 mM MgClzm, 0.1 mM 211012,
`1 mM
`spermidine, (10 units for 15 minutes at 37°C followed by 15 minutes at 58° C), phenol and chloroform
`extracted, ethanol precipitated and resuspended to 250 ng/ul in TE buffer.
`Twenty nanograms of the pG fragment and 25 ng of the pUC18 fragment were ligated overnight at
`15' C in a 20 ul volume. One hundred microiiters of competent DH5a cells were transformed with a portion
`of the ligation mixture. Colonies were screened for the presence of a single Pst l site, which is within the
`SV40 region of pG. There is an additional Pst | site in the ampr gene of pBR322 but not in the ampr gene of
`pUC18. The plasmids were also screened for the regeneration of the EcoR | site upon ligation of Pvu ll
`ends with filled-in EccR 1 ends. The resultant plasmid was termed pUCG.
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`10
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`15
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`20
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`25
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`30
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`35
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`45
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`50
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`55
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`iii. pUCG to pUCG-MCSil (Figs. 1b and 10)
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`18
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`1r;
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`I)
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