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Cloning and characterization of a new gene involved in lymphocyte activation

Meng Zhang, University of Massachusetts Amherst


Cattle represent an economically important species whose immune system seems to depart from the standard human and murine models. Little has been documented about bovine lymphocyte activation in vitro, such work is needed for comparative immunology and for us to understand bovine immunology. In general, lymphocyte activation is accompanied by many molecular changes at the mRNA level. The unique characteristics of lymphocyte activation imply that a unique set of genes is associated with this biological process. However, there is very little known (in any species) about lymphocyte-specific genes that are differentially up-regulated after activation. In this thesis, bovine lymphocyte activation was first stimulated in vitro. Secondly, representational difference analysis (RDA) method was used to clone mRNAs that are exclusively present in activated bovine lymphocytes. Subsequently, the cDNAs were analyzed by DNA sequencing and homology search against the Genbank database. Clone E8 was identified as a potential G-protein-coupled receptor. E8 is up-regulated in activated bovine lymphocytes at 2 hours post stimulation. When up-regulated, E8 mRNA level remains constant from 2 hours to at least 72 hours post stimulation. Similar kinetic expression of E8 is observed following either LPS or Con A stimulations. Expression of E8 was also detected in murine lymphocytes upon activation with LPS or Con A and with similar kinetic expression. E8 showed increased level of expression when human T and B lymphocytes were activated by cross-linking of antigen receptors along with costimulatory molecules. E8 expression was found not to be associated with resting non-lymphoid tissues, activated non-lymphoid cell lines, nor activated macrophages and neutrophils. Therefore, E8 represents an early gene specific to lymphocyte activation. The size of the full-length transcript of clone E8 was estimated at about 2.2 kb. A full-length cDNA was obtained by the RACE procedure. Sequence alignment revealed that E8 is homologous to EB11, a human gene induced by EBV; CXCR1, as well as human CCR4 and CCR5 genes. Potential biological functions of this gene are discussed.

Subject Area

Molecular biology|Veterinary services|Cellular biology|Immunology

Recommended Citation

Zhang, Meng, "Cloning and characterization of a new gene involved in lymphocyte activation" (1997). Doctoral Dissertations Available from Proquest. AAI9809416.