This study investigated the biological activity of a macrophage activating factor (MAF) produced by activated lymphocytes from the rainbow trout (Oncorhynchus mykiss) and attempts to discover its molecular source. Peripheral blood lymphocytes were shown to release factors with MAF activity following incubation with a variety of stimulants and were subsequently shown to activate macrophages using at least two different methods, the nitroblue tetrazolium (NBT) colourimetric assay and the luminol-dependent chemiluminescent assay. The latter technique detected an immediate response which decayed over a 40 minute period on the addition of cell-free supernatants from activated lymphocytes to macrophages. A number of molecular approaches, including degenerate PCR primer amplification, DNA cross-hybridisation and cDNA library screening were used in this study to try to isolate any cytokine genes from Oncorhynchus mykiss. As a control β-actin cDNA was successfully amplified from Oncorhynchus mykiss using primers based on the salmon sequence. The Oncorhynchus mykiss orthologue of IFN-y was initially targeted. However, although a PCR product of the appropriate size was amplified using degenerate primers based on mammalian and avian IFN-y sequences, the sequence was not related to IFN-y or any other known Oncorhynchus mykiss sequence. A similar strategy was used to try and amplify the Oncorhynchus mykiss orthologue of mammalian IL-15. Again despite amplification of a DNA fragment of approximately the correct size there appeared to be no relationship between it and the known IL-15 sequences. As an alternative strategy a cDNA library from stimulated peripheral blood lymphocytes (PBLs) was constructed and screened using cDNA probes derived from stimulated and non-stimulated PBLs in order to detect mRNAs which might have been upregulated as a result of in vitro stimulation. A number of positive clones were obtained from the differential screening of the library including cDNAs showing similarity to other unidentified fish sequences as well as to a number of proteins predicted to be involved in regulation of cell proliferation, neocorticogenesis and embryo development. Additionally. the library was also screened using ovine cytokine cDNA probes. although no positively hybridising clones were obtained. The ovine IFN-y gene was also used to probe genomic DNA from Oncorhynchus mykiss. but unlike previous studies with human IFN-y gene no hybridisation between the ovine IFN-y gene and Oncorhynchus mykiss DNA was observed. This investigation highlights the potential difficulties of using various molecular strategies such as DNA cross-hybridisation or PCR techniques for the cloning of fish cytokine sequences. Consequently, future strategies for cloning fish cytokine genes may require targeting the biological activity through expression libraries.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:403338 |
| Date | January 2003 |
| Creators | Sharif, Rubina Qasour |
| Publisher | University of Stirling |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1893/26687 |
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