Genetic strategies for analyzing proteins: Applications utilizing the R388 type II dihydrofolate reductase

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dc.contributor.advisorBennett, George N.en_US
dc.creatorVermersch, Polly Smithen_US
dc.date.accessioned2009-06-03T23:58:29Zen_US
dc.date.available2009-06-03T23:58:29Zen_US
dc.date.issued1988en_US
dc.description.abstractApplications of recombinant DNA technology to protein analysis have been demonstrated using the trimethoprim resistant (Tp$\sp{\rm R}$)R388 type II dihydrofolate reductase (DHFR). DHFR fusion vectors were constructed which contain cloning sites in the C-terminal coding region of the DHFR. Segments of nematode major sperm protein (MSP) fused to the DHFR were recognized by antibody to MSP. A 15 aa segment of MSP sequence, fused to the DHFR, was shown to be sufficient to elicit antibody to MSP. A two-step procedure was developed for purifying the DHFR proteins. A selectable FokI/supF cassette was developed to facilitate DNA excision and replacement mutagenesis. When the cassette is inserted into DNA, the presence of the tyrosine tRNA suppressor gene (supF) contained on the cassette is selected by the suppression of amber mutations in the recipient host. Subsequent refined mutagenesis is possible due to the unique cleavage properties of FokI. The cassette/vector system was used to produce a deletion corresponding to amino acid residues 2-7 of the DHFR which did not noticeably impair Tp$\sp{\rm R}$. Resistance was abolished, however, by a deletion of amino acid residues 2-21. A structural gene was synthesized which contains many unique restriction sites and encodes a Tp$\sp{\rm R}$ DHFR which is 10 aa shorter at the N-terminus relative to natural type II DHFRs. High copy number vectors which utilize strong promoters to transcribe the dhfr gene and the primer for plasmid replication were constructed to overproduce the mini DHFR and a full-length derivative. Site-directed mutagenesis was used to test the importance of glu-58 and thr-48 in a putative folate binding site of the mini and full-length DHFRs. The introduction of thr-58 and/or glu-48 destroyed in vivo function of the DHFRs. Tp$\sp{\rm R}$ was retained in the full length gln-58 R388 DHFR, but not in the mini gln-58 DHFR. Through random mutagenesis a mini Tp$\sp{\rm R}$gln-58 R388 DHFR was obtained that contained a duplication of leu-pro-ser, at the N-terminus. Successive additions of the leu-pro-ser triplet to the N-terminus appear to stabilize the functional form of the mini DHFR.en_US
dc.format.extent241 p.en_US
dc.format.mimetypeapplication/pdfen_US
dc.identifier.callnoThesis Biochem. 1988 Vermerschen_US
dc.identifier.citationVermersch, Polly Smith. "Genetic strategies for analyzing proteins: Applications utilizing the R388 type II dihydrofolate reductase." (1988) Diss., Rice University. <a href="https://hdl.handle.net/1911/16196">https://hdl.handle.net/1911/16196</a>.en_US
dc.identifier.urihttps://hdl.handle.net/1911/16196en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectBiochemistryen_US
dc.titleGenetic strategies for analyzing proteins: Applications utilizing the R388 type II dihydrofolate reductaseen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
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