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Title: The isolation and characterisation of a mouse C1 inhibitor cDNA : its use in RNA and protein expression studies
Author: Russell, Jacqueline Anne
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1996
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C1 inhibitor is expressed primarily by the liver but also by peripheral cells such as monocytes. In primary human monocytes C1 inhibitor mRNA stability is regulated by interferon-gamma. However, these cells did not express C1 inhibitor within the first 24 hours after isolation unless stimulated with interferon-gamma. Therefore, there was no control for the study of mRNA stability. The human monocyte-like cell lines THP1 and U937 did not express C1 inhibitor mRNA even following interferon-gamma treatment. As an alternative, the mouse cell line J774 was investigated. For this work, a mouse. C1 inhibitor cDNA was isolated. Using this mouse sequence as a probe showed that J774 cells do not express C1 inhibitor and so were not suitable models for further study. Mouse C1 inhibitor cDNA was isolated using a mouse liver cDNA library, 5' rapid amplification of cDNA ends and reverse transcription-polymerase chain reaction. The transcript consisted of 1795 nucleotides which can be translated to give an open reading frame of 505 amino acids, predicted molecular weight 55.5 KDa. The sequence is available from EMBL, accession number Y10386. A comparison between mouse, human and bovine C1 inhibitor showed 65.1% identity between the three proteins. Each can be divided into three parts. The signal peptide which is highly conserved, the amino terminal extension which is divergent at the primary sequence level but is predicted to contain multiple sites for O-glycosylation, and the serpin domain which is also conserved. The serpin domain of mouse C1 inhibitor was expressed using an in vitro transcription-translation system. It was shown to have inhibitory activity against human C1s, demonstrating that the sequence differences between mouse and human C1 inhibitor do not crucially affect this aspect of their function in vitro. The human and mouse C1 inhibitor serpin domains were modelled on previously determined serpin structures. The sequence differences could easily be accommodated into a similar three dimensional structure.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available