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UBC Theses and Dissertations

Characterization of a murine activated lymphocyte antigen (MALA-2) : the murine homologue of human intercellular adhesion molecule 1 (ICAM-1) Horley, Kathleen J.

Abstract

A previously characterized rat monoclonal antibody, YN1/1.7, recognizes an antigen termed murine activated lymphocyte antigen (MALA-2). MALA-2 is a 95-100 kD monomeric glycoprotein and is expressed on mitogen activated spleen cells but is present at low levels onthymocytes, fibroblasts, and lymph node cells. Interestingly, YN1/1.7, inhibits mixed lymphocyte reaction (MLR) suggesting that MALA-2 is directly involved in lymphocytea ctivation. In this research project, the gene encoding MALA-2 was characterized by cDNA cloning, genomic cloning, and analysis of an assumed alternatively spliced mRNA. Two cDNA clones were isolated from an NS-1 cDNA library using oligonucleotide probes constructed from amino acid sequences of peptides derived by tryptic cleavage. The two cDNAs, 1(4-1.1 and 1(3-1.1, both encode MALA-2 but differ in their 5' untranslated sequences and those encoding the leader and N-terminal nine amino acids. MALA-2 is a transmembrane glycoprotein with five immunoglobulin-like domains. It displays homology with the human intercellular adhesion molecule 1 (ICAM-1), as well as human ICAM-2, human ICAM-3, and murine ICAM-2. Screening of genomic libraries yielded a partial genomic clone (4.0 kb ), containing five 3'exons and a pseudo exon. The five exons are common to both cDNAs and have consensus splice donor and acceptor sequences. The pseudo exon lacks these splice donor and acceptor sequences. The exons encoding the 5 region of K4-1.1 were not isolated, but using data from Southern blot analyses a proposed map of the whole gene was constructed. Two 4.0 kb Barn HI fragments seem to contain all of the exons, with the 5' region probably consisting of two exons, and being located at least 6.0 kb upstream of the five 3' exons. The 1(3-1.1 cDNA has not been reported elsewhere, thus it was further analysed for its authenticity. The 5' region unique to1(3-1.1 did not seem to be linked to the 5' region of K4-1.1, and Northern blot analysis failed to detect a 3.0 kb message corresponding to the K3-1.1 cDNA. However, PCR analysis using primers spanning the common junction between the two cDNAs detected a fragment. These data suggest that a K3-1.1 transcript can exist but may be expressed at a very low level.

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