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The Organization and Expression of the Human Dihydrofolate Reductase Gene in Methotrexate-Resistant and Methotrexate-Sensitive Cell Lines

Citation

Masters, Jeffrey Nelson (1985) The Organization and Expression of the Human Dihydrofolate Reductase Gene in Methotrexate-Resistant and Methotrexate-Sensitive Cell Lines. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/jaa8-jx74. https://resolver.caltech.edu/CaltechTHESIS:01312019-142248860

Abstract

The analysis of several derivatives of the human cell lines HeLa and VA2-B, selected for resistance to methotrexate (MTX), shows a striking variability in the dihydrofolate reductase (DHFR) enzyme levels, chromosome constitution and growth characteristics in the absence of MTX. In contrast to the mouse system, the number of double minutes of the human cells does not correlate with either the increased DHFR levels or the instability of the amplified phenotype.

The isolation of human DHFR eDNA by differential hybridization or by phenotypic expression in E. coli, facilitates the characterization of the human DHFR coding region and its multiple mRNAs. Comparison of the DNA sequences of several DHFR cDNAs shows a high degree of homology between the coding regions of the human and mouse genes (89%) with no obvious identity in the 3'-untranslated region. The analysis also demonstrates that cDNAs of the 3 identified mRNAs are colinear in the 3'-end sequence, and that polyadenylation occurs at different sites.

Hybridization of EcoRI digested nuclear DNA from the MTX-resistant 6A3 cells with DHFR cDNAs shows EcoRI fragments that are either unamplified or amplified relative to the same fragments of human sperm, HeLa cell, and VA2-B cell DNA. Two of the unamplified fragments were isolated from a cosmid library made from human sperm DNA. The DNA sequence analysis shows that these two fragments contain a DHFR intronless pseudogene including the EcoRI site found in the DHFR cDNAs. If an RNA intermediate directs the formation of this pseudogene, an RNA larger than the 1.0 kb DHFR mRNA must be involved. In contrast to the unamplified DNA fragments, the amplified fragments contain the exons of the human DHFR gene. The gene is about 29 kb in length, with five introns interrupting the DHFR coding region in the same positions found in the mouse gene. The DHFR mRNAs were mapped as a major 5'-end at position -71 of the human DHFR gene. In addition, six minor 5'-ends of DHFR-specific polysomal RNA were mapped from positions -449 to -480 and represent about 1% of the major transcripts. The upstream transcripts are relatively enriched in the nuclear RNA fraction, indicating a different regulation of expression for these minor transcripts.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Molecular Biology
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Molecular Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Attardi, Giuseppe
Thesis Committee:
  • Campbell, Judith L. (chair)
  • Davidson, Norman R.
  • Lewis, Edward B.
  • Meyerowitz, Elliot M.
  • Attardi, Giuseppe
Defense Date:24 July 1984
Funders:
Funding AgencyGrant Number
Earle C. Anthony FellowshipUNSPECIFIED
NIHUNSPECIFIED
Jean Weigle Memorial FundUNSPECIFIED
Record Number:CaltechTHESIS:01312019-142248860
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:01312019-142248860
DOI:10.7907/jaa8-jx74
Related URLs:
URLURL TypeDescription
https://doi.org/10.1128/mcb.2.5.498DOIArticle adapted for Ch. 2
https://doi.org/10.1016/0022-2836(82)90268-6DOIArticle adapted for Ch. 3
https://doi.org/10.1016/0378-1119(83)90147-6DOIArticle adapted for Ch. 4
https://doi.org/10.1016/s0022-2836(83)80032-1DOIArticle adapted for Ch. 5
https://doi.org/10.1016/0022-2836(84)90419-4DOIArticle adapted for Ch. 6
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11372
Collection:CaltechTHESIS
Deposited By: Mel Ray
Deposited On:01 Feb 2019 19:35
Last Modified:16 Apr 2021 22:21

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