The CD45 glycoprotein family exhibits cell line-age-associated structural heterogeneity arising in part from alternate 5' exon shuffling. Previous studies of exons involved in the final glycoprotein structure have provided evidence of alternate exon use for only three exons (Ex-4, 5 and 6). However, our prior data using reverse transcription-polymerase chain reaction (RT-PCR) suggested the presence of at least one additional CD45 alternate exon. By using RT-PCR, Southern blotting with exon-specific or exon splice junction-specific oligonucleotide probes and direct DNA sequencing of RT-PCR products, we demonstrated additional alternate use involving Ex-7. PCR analysis of stage I thymocytes (CD4-CD8-) revealed only faintly detectable bands for two isoforms: one lacking Ex-4, 5, 6 and 7 (a "minus-one" [Ex(-1)] isoform), and a smaller isoform preliminarily characterized as also lacking Ex-8. Stage II thymocytes (CD4+CD8+) prominently expressed both Ex(-1) and zero alternate exon (Ex(0] isoforms, with one exon (Ex(1] and two exon (Ex(2] isoforms also present. Among stage III thymocytes, both CD4+CD8- and CD4-CD8+ cells expressed only Ex(-1) and Ex(0) isoforms. CD45 alternate exon use in resting CD4+ and CD8+ lymph node T cells was divergent, with CD8+ cells additionally expressing an Ex(2) isoform. Among alloreactive T cell clones, band intensity for the Ex(1) isoform in CD4+ BC-3 cells was much less than for resting CD4+ T cells, while the CD8+ CTL clone 8.2.2 exhibited production of the higher alternate exon isoforms, Ex(2) and Ex(3). We conclude that at least four and possibly five alternate exons exist in the CD45 glycoprotein family, with a previously unrecognized isoform lacking Ex-4, 5, 6 and 7 prominently expressed in T cells. Shuffling of CD45 alternate exons appears to occur in an organized and predictable sequence during cellular maturation and activation.

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