Karoly Fatyol

Abstract

In this study we have analyzed the activity of the silkworm A3 cytoplasmic actin promoter in homologous insect and heterologous mammalian expression systems. To study the expression of the A3 actin promoter in insect cells we have isolated and characterized a stably transformed Bombyx mori cell line containing a novel selectable marker gene, puromycin N-acetyl transferase under control of transcriptional regulatory signals from the A3 cytoplasmic actin gene. Integration of the transfected plasmids into the host genome was demonstrated by Southern and in situhybridizations. By using this cell line we have identified alternative transcriptional initiation sites for the A3 cytoplasmic actin gene. One of these start sites is located ~35 bp upstream from the earlier determined transcription initiation site. The two mRNA start sites are utilized with a similar efficiency in the BmN cell line. In addition we detected transcripts that initiated within the first intron of the A3 actin gene. These transcripts may be synthesized under control of an alternative promoter. The mRNA isoforms generated by the use of the alternative start sites may exhibit different characteristics (stability etc.), therefore the alternative promoter and initiation site usage may be an important factor in regulating the cyclic changes of the A3 actin mRNA level in the silk gland during larval morphogenesis.

Here we also report that A3 cytoplasmic actin based vectors can express foreign genes in various vertebrate cells. Using an A3 actin based expression cassette we have isolated stably transformed mammalian cell lines. Isolation of transformed cells was based on expression of the selected marker gene. Stable integration of A3 actin plasmid construct into the host genome was demonstrated. The integrated plasmid molecules frequently exhibited amplification which may indicate the presence of amplification promoting elements in the 5' or 3' UTR of the A3 actin gene. This is the first reported case for the use of an insect promoter driven selection cassette to establish transformed mammalian cell lines.

The regulatory elements of the A3 actin 5' UTR that are necessary for the expression in mammalian cells were studied by transient transfection assay. We demonstrated that the earlier identified promoter elements of the A3 actin gene are not required for transcriptional activity in mammalian cells. On the contrary, the A3 actin first intron contains a strong promoter that exhibits a transcriptional activity in mouse cells that is comparable to the activity of the strong SV40 early promoter. We suggest that the identified intronic promoter is the same promoter region that regulates the synthesis of the rare A3 actin mRNA isoform observed in Bombyx mori cells

LLU Discipline

Biochemistry

Department

Biochemistry

School

Graduate School

First Advisor

Aladar A. Szalay

Second Advisor

Alan Escher

Third Advisor

Thomas A. Linkhart

Fourth Advisor

John F. Sands

Fifth Advisor

Charles W. Slattery

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1998

Date (Title Page)

6-1998

Language

English

Library of Congress/MESH Subject Headings

Microfilament Proteins; Genes, Regulators; Actins -- genetics; Reverse Transcriptions; Enchancer Elements (Genetics)

Type

Dissertation

Page Count

x; 142

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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