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A Study of a Temperature-Sensitive Chinese Hamster Cell Mutant with a Defect in Rna Metabolism.

Record Type:	Language materials, manuscript : Monograph/item
Title/Author:	A Study of a Temperature-Sensitive Chinese Hamster Cell Mutant with a Defect in Rna Metabolism./
Author:	Wong, Eric Alan.
Description:	1 online resource (171 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 42-07, Section: B.
Contained By:	Dissertations Abstracts International42-07B.
Subject:	Biology. -
Online resource:	click for full text (PQDT)
ISBN:	9798661745651

A Study of a Temperature-Sensitive Chinese Hamster Cell Mutant with a Defect in Rna Metabolism.
Wong, Eric Alan.

A Study of a Temperature-Sensitive Chinese Hamster Cell Mutant with a Defect in Rna Metabolism. - 1 online resource (171 pages)

Source: Dissertations Abstracts International, Volume: 42-07, Section: B.

Thesis (Ph.D.)--University of California, San Diego, 1981.

Includes bibliographical references

This thesis describes the isolation and study of a new temperature-sensitive mutant of Chinese hamster fibroblasts. Following a shift from the permissive temperature (34(DEGREES)C) to the non-permissive temperature (40.5(DEGREES)C), mutant cells rapidly lost viability, and the rates of DNA, RNA, and protein synthesis also declined rapidly and in parallel. The experiments described attempt to biochemically define the primary defect in the mutant. A general metabolic defect affecting cellular energy metabolism was investigated, but neither the rate of respiration in whole cells nor the energy charge was altered in the mutant at 40.5(DEGREES)C. In mutant cells synchronized in early G(,1), the rates of RNA and protein synthesis at 40.5(DEGREES)C declined before the induction of DNA synthesis, indicating that the effect on DNA synthesis was secondary. Therefore, a detailed investigation of protein synthesis was conducted. The effects of the temperature shift on the protein biosynthetic machinery were studied in vivo by an analysis of polysome profiles, a measurement of transit times, and an analysis of ('35)S-met-tRNA binding to 40S ribosomal subunits. In addition, protein synthesis in cell free extracts (S30s) was examined using the endogenous mRNAs, or in S30 extracts made messenger RNA dependent by the action of a Ca('+2) activated nuclease. A synthetic mRNA (polyuridylic acid) or natural mRNAs (rabbit (beta)-globin mRNA or VSV in vitro transcripts) were utilized as exogenous sources of mRNA to determine the capacities of the S30 extracts for translation. The results of all of these experiments suggested that neither the initiation, elongation, or termination steps of protein synthesis were affected; but that the temperature-sensitive defect altered the cytoplasmic levels of mRNA. This led us to focus our attention on RNA synthesis. An investigation of RNA synthesis and metabolism in the mutant cells at 40.5(DEGREES)C revealed that both total poly(A)('+) and poly(A)('-) RNA synthesis were reduced by 50%, 12 hours after the temperature shift. The syntheses of rRNA and mRNA were affected most, while the synthesis of small RNAs was relatively unchanged. An analysis of the RNA in the cytoplasm showed that the levels of the stable rRNAs were unchanged, while the level of the more labile mRNA was decreased to half of that at 34(DEGREES)C. Those mRNAs present in the mutant at 40.5(DEGREES)C were capable of directing protein synthesis in the rabbit reticulocyte system with the same efficiency as mRNA from cells at 34(DEGREES)C. Finally, the effect of the temperature shift on the synthesis of the Mr 69,000 heat shock protein was investigated in mutant cells. The induction of this protein (p69) was shown to require new mRNA synthesis, and the message for p69 was shown to possess a very short functional half life. The synthesis of p69 was induced within 3 hours of the temperature shift but became undetectable by 12 hours at 40.5(DEGREES)C in the mutant, whereas in the wild type cells its synthesis continued. This inhibition of p69 synthesis in the mutant at 40.5(DEGREES)C, could be mimicked in wild type cells by blocking new mRNA synthesis after the three hour induction period with the drug dichloro-ribofuranosylbenzimidazole (DRB). This implied that the loss of the capacity to synthesize p69 in the mutant at 40.5(DEGREES)C was due to a loss of the mRNA for p69 from turnover and a block in the production of new mRNA.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2024

Mode of access: World Wide Web

ISBN: 9798661745651Subjects--Topical Terms:

599573
Biology.
Index Terms--Genre/Form:

554714
Electronic books.

A Study of a Temperature-Sensitive Chinese Hamster Cell Mutant with a Defect in Rna Metabolism.
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100 1 $a Wong, Eric Alan. $3 1466574
245 1 2 $a A Study of a Temperature-Sensitive Chinese Hamster Cell Mutant with a Defect in Rna Metabolism.
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300 $a 1 online resource (171 pages)
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500 $a Source: Dissertations Abstracts International, Volume: 42-07, Section: B.
500 $a Publisher info.: Dissertation/Thesis.
502 $a Thesis (Ph.D.)--University of California, San Diego, 1981.
504 $a Includes bibliographical references
520 $a This thesis describes the isolation and study of a new temperature-sensitive mutant of Chinese hamster fibroblasts. Following a shift from the permissive temperature (34(DEGREES)C) to the non-permissive temperature (40.5(DEGREES)C), mutant cells rapidly lost viability, and the rates of DNA, RNA, and protein synthesis also declined rapidly and in parallel. The experiments described attempt to biochemically define the primary defect in the mutant. A general metabolic defect affecting cellular energy metabolism was investigated, but neither the rate of respiration in whole cells nor the energy charge was altered in the mutant at 40.5(DEGREES)C. In mutant cells synchronized in early G(,1), the rates of RNA and protein synthesis at 40.5(DEGREES)C declined before the induction of DNA synthesis, indicating that the effect on DNA synthesis was secondary. Therefore, a detailed investigation of protein synthesis was conducted. The effects of the temperature shift on the protein biosynthetic machinery were studied in vivo by an analysis of polysome profiles, a measurement of transit times, and an analysis of ('35)S-met-tRNA binding to 40S ribosomal subunits. In addition, protein synthesis in cell free extracts (S30s) was examined using the endogenous mRNAs, or in S30 extracts made messenger RNA dependent by the action of a Ca('+2) activated nuclease. A synthetic mRNA (polyuridylic acid) or natural mRNAs (rabbit (beta)-globin mRNA or VSV in vitro transcripts) were utilized as exogenous sources of mRNA to determine the capacities of the S30 extracts for translation. The results of all of these experiments suggested that neither the initiation, elongation, or termination steps of protein synthesis were affected; but that the temperature-sensitive defect altered the cytoplasmic levels of mRNA. This led us to focus our attention on RNA synthesis. An investigation of RNA synthesis and metabolism in the mutant cells at 40.5(DEGREES)C revealed that both total poly(A)('+) and poly(A)('-) RNA synthesis were reduced by 50%, 12 hours after the temperature shift. The syntheses of rRNA and mRNA were affected most, while the synthesis of small RNAs was relatively unchanged. An analysis of the RNA in the cytoplasm showed that the levels of the stable rRNAs were unchanged, while the level of the more labile mRNA was decreased to half of that at 34(DEGREES)C. Those mRNAs present in the mutant at 40.5(DEGREES)C were capable of directing protein synthesis in the rabbit reticulocyte system with the same efficiency as mRNA from cells at 34(DEGREES)C. Finally, the effect of the temperature shift on the synthesis of the Mr 69,000 heat shock protein was investigated in mutant cells. The induction of this protein (p69) was shown to require new mRNA synthesis, and the message for p69 was shown to possess a very short functional half life. The synthesis of p69 was induced within 3 hours of the temperature shift but became undetectable by 12 hours at 40.5(DEGREES)C in the mutant, whereas in the wild type cells its synthesis continued. This inhibition of p69 synthesis in the mutant at 40.5(DEGREES)C, could be mimicked in wild type cells by blocking new mRNA synthesis after the three hour induction period with the drug dichloro-ribofuranosylbenzimidazole (DRB). This implied that the loss of the capacity to synthesize p69 in the mutant at 40.5(DEGREES)C was due to a loss of the mRNA for p69 from turnover and a block in the production of new mRNA.
533 $a Electronic reproduction. $b Ann Arbor, Mich. : $c ProQuest, $d 2024
538 $a Mode of access: World Wide Web
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655 7 $a Electronic books. $2 local $3 554714
690 $a 0306
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710 2 $a University of California, San Diego. $3 1187935
773 0 $t Dissertations Abstracts International $g 42-07B.
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