Molecular cloning and expression of the mammalian basic fibroblast growth factor antisense mRNA.
Date
1997
Authors
Knee, Rai.
Journal Title
Journal ISSN
Volume Title
Publisher
Dalhousie University
Abstract
Description
The fibroblast growth factor (FGF-2) gene is bidirectionally transcribed wherein the sense DNA strand yields multiple mRNA species responsible for encoding FGF-2 protein, and the antisense strand generates a single 1.5 kb mRNA. Formation of double-stranded complexes between sense and antisense mRNAs has been suggested as a posttranscriptional mechanism in the regulation of FGF-2 gene expression. The majority of this thesis describes the cloning and characterization of the rat FGF-2 antisense (FGF-AS) mRNA.
Northern and RT-PCR analyses of various rat and human tissues reveal that the AS mRNA is expressed in a tissue-specific and developmentally regulated fashion. A neonatal rat liver library was constructed for the isolation of a full-length rat FCF-AS cDNA. Sequence analysis reveals that the rat FGF-AS transcript is a 1.1 kb polyadenylated RNA with a conserved ORF that specifies a novel 35 kDa protein. The rat AS and sense mRNAs contain 425 bp of complementary overlap at their 3$\sp\prime$ regions. Inverse levels of FGF-2 sense and AS mRNA observed in brain, liver, and certain tumor cell lines, are consistent with the possibility of AS regulation of FGF-2 mRNA.
The FGF-AS mRNA encodes a 35 kDa protein which is a novel member of the family of MutT/nudix hydrolyases. This diverse family of enzymes includes the antimutator 8-oxo-dGTPases which hydrolyze mutagenic 8-oxo-dCTP and thereby suppress the occurrence of spontaneous oxidative mutagenesis. Sequence-specific antibodies recognized the in vitro translated antisense protein and detected a 35 kDa protein in Western blots of rat and mouse tissues. FGF-AS mRNA expression in rat glioma cells was shown to increase in response to hydrogen peroxide-induced oxidative stress. The abundant expression of FGF-AS in neonatal liver and tissues such as adrenal, kidney and heart, suggests that the functional role of the antisense protein is likely related to the high metabolic function and/or oxidative stress inherent to these tissues.
Thesis (Ph.D.)--Dalhousie University (Canada), 1997.
Northern and RT-PCR analyses of various rat and human tissues reveal that the AS mRNA is expressed in a tissue-specific and developmentally regulated fashion. A neonatal rat liver library was constructed for the isolation of a full-length rat FCF-AS cDNA. Sequence analysis reveals that the rat FGF-AS transcript is a 1.1 kb polyadenylated RNA with a conserved ORF that specifies a novel 35 kDa protein. The rat AS and sense mRNAs contain 425 bp of complementary overlap at their 3$\sp\prime$ regions. Inverse levels of FGF-2 sense and AS mRNA observed in brain, liver, and certain tumor cell lines, are consistent with the possibility of AS regulation of FGF-2 mRNA.
The FGF-AS mRNA encodes a 35 kDa protein which is a novel member of the family of MutT/nudix hydrolyases. This diverse family of enzymes includes the antimutator 8-oxo-dGTPases which hydrolyze mutagenic 8-oxo-dCTP and thereby suppress the occurrence of spontaneous oxidative mutagenesis. Sequence-specific antibodies recognized the in vitro translated antisense protein and detected a 35 kDa protein in Western blots of rat and mouse tissues. FGF-AS mRNA expression in rat glioma cells was shown to increase in response to hydrogen peroxide-induced oxidative stress. The abundant expression of FGF-AS in neonatal liver and tissues such as adrenal, kidney and heart, suggests that the functional role of the antisense protein is likely related to the high metabolic function and/or oxidative stress inherent to these tissues.
Thesis (Ph.D.)--Dalhousie University (Canada), 1997.
Keywords
Biology, Molecular.