The objectives of this research were to characterize insulin-like growth factor-I (IGF-I) gene expression in cattle, to determine how IGF-I gene expression is affected by nutritional intake and growth hormone (GH) in cattle, and to identify the regulatory DNA region that mediates GH stimulation of IGF-I gene expression. It was found that transcription of the IGF-I gene in cattle was initiated from both exon 1 and exon 2, generating class 1 and class 2 IGF-I mRNA, respectively. Both classes of IGF-I mRNA appeared to be ubiquitously expressed, with the highest level in liver and with class 1 being more abundant than class 2 in all tissues examined. Class 1 IGF-I mRNA may be also translated more efficiently than class 2 IGF-I mRNA. Liver expression of IGF-I mRNA was decreased (P < 0.01) by food deprivation in cattle, and this decrease was due to an equivalent decrease in both classes of IGF-I mRNA. Liver expression of IGF-I mRNA was increased (P < 0.01) by GH, and this increase resulted mainly from increased expression of class 2 IGF-I mRNA. Using cotransfection analyses, a ~700 bp chromosomal region ~75 kb 5' from the first exon of the human IGF-I gene was found to enhance reporter gene expression in the presence of constitutively active signal transducer and activator of transcription 5 (STAT5) proteins, transcription factors that are known to be essential for GH-increased IGF-I gene expression. This 700 bp DNA region contains two STAT5-binding sites that appear to be conserved in mammals including cattle. Electrophoretic mobility shift assays and cotransfection analyses confirmed their ability to bind to STAT5 proteins and to mediate STAT5 activation of gene expression, respectively. Chromatin immunoprecipitation assays indicated that overexpressed constitutively active STAT5b protein bound to the chromosomal region containing these two STAT5-binding sites in Hep G2 cells, and this binding was associated with increased expression of IGF-I mRNA. These two STAT5-binding sites were also able to mediate GH-induced STAT5 activation of gene expression in reconstituted GH-responsive cells. These results together suggest that the distal DNA region that contains two STAT5-binding sites may mediate GH-induced STAT5 activation of IGF-I gene transcription in vivo. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/30208 |
| Date | 30 December 2005 |
| Creators | Wang, Ying |
| Contributors | Animal and Poultry Sciences, Jiang, Honglin, Wong, Eric A., Smith, Edward J., Webb, Kenneth E. Jr., Akers, Robert Michael |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | dissertation.pdf |
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