Changes in dihydrofolate reductase (DHFR) mRNA levels can account fully for changes in DHFR synthesis rates during terminal differentiation in a highly amplified myogenic cell line

E E Schmidt; G F Merrill

doi:10.1128/MCB.11.7.3726

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Changes in dihydrofolate reductase (DHFR) mRNA levels can account fully for changes in DHFR synthesis rates during terminal differentiation in a highly amplified myogenic cell line

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Changes in dihydrofolate reductase (DHFR) mRNA levels can account fully for changes in DHFR synthesis rates during terminal differentiation in a highly amplified myogenic cell line

E E Schmidt and G F Merrill

Molecular and cellular biology, Vol.11(7), pp.3726-3734

07/1991

DOI: https://doi.org/10.1128/MCB.11.7.3726

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https://hdl.handle.net/2376/116972

PMCID: PMC361140

PMID: 2046674

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https://doi.org/10.1128/MCB.11.7.3726View

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Abstract

Blotting, Southern

Cell Line

RNA, Messenger - genetics

Tetrahydrofolate Dehydrogenase - isolation & purification

Enzyme Induction

Ribosomes - metabolism

RNA, Messenger - metabolism

Tetrahydrofolate Dehydrogenase - biosynthesis

DNA - genetics

Animals

Gene Amplification

Muscles - enzymology

Polyribosomes - metabolism

Cell Differentiation

Mice

Kinetics

Muscles - cytology

Tetrahydrofolate Dehydrogenase - genetics

Dihydrofolate reductase (DHFR) enzyme is preferentially synthesized in proliferative cells. A mouse muscle cell line resistant to 300 microM methotrexate was developed to investigate the molecular levels at which DHFR is down-regulated during myogenic withdrawal from the cell cycle. H- alpha R300T cells contained 540 copies of the endogenous DHFR gene and overexpressed DHFR mRNA and DHFR protein. Despite DHFR gene amplification, the cells remained diploid. As H- alpha R300T myoblasts withdrew from the cell cycle and committed to terminal differentiation, DHFR mRNA levels and DHFR synthesis rates decreased with closely matched kinetics. After 15 to 24 h, committed cells contained 5% the proliferative level of DHFR mRNA (80 molecules per committed cell) and synthesized DHFR protein at 6% the proliferative rate. At no point during the commitment process did the decrease in DHFR synthesis rate exceed the decrease in DHFR message. The decrease in DHFR mRNA levels during commitment was sufficient to account fully for the decrease in rates of DHFR synthesis. Furthermore, DHFR mRNA remained polysomal, and the average number of ribosomes per message remained constant (five to six ribosomes per DHFR mRNA). The constancy of polysome size, along with the uniform rate of DHFR synthesis per message, indicated that DHFR mRNA was efficiently translated in postreplicative cells. The results support a model wherein replication-dependent changes in DHFR synthesis rates are determined exclusively by changes in DHFR mRNA levels.

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Title: Changes in dihydrofolate reductase (DHFR) mRNA levels can account fully for changes in DHFR synthesis rates during terminal differentiation in a highly amplified myogenic cell line
Creators: E E Schmidt - Department of Biochemistry and Biophysics, Oregon State University, Corvallis
G F Merrill
Publication Details: Molecular and cellular biology, Vol.11(7), pp.3726-3734
Academic Unit: UNKNOWN
Publisher: United States
Grant note: AG00334 / NIA NIH HHS GM34432 / NIGMS NIH HHS
Identifiers: 99900548469701842
Language: English
Resource Type: Journal article