Similarity of PAGE-1, GAGE, and MAGE. (A) The predicted PAGE-1 reading frame is derived from the full-length PAGE-1 EST clone nh32c06. The GAGE and MAGE sequences are from SwissProt: GGE1, GGE2, GGE3, GGE4, GGE5, GGE6, MAG5, and MAG8_HUMAN. Note that the “MAGE alignment” matches amino acids that occur in MAGE5 and/or MAGE8, which are similar to PAGE-1 and/or GAGE1–6; the homologies between single members of the MAGE and PAGE and GAGE protein families are weaker. (B) Alignment of PAGE-1 with other PAGEs. PAGE-2 was translated from the EST ai61a04 EST cluster and PAGE-3 from om29f08. PAGE-3 was translated from one single EST and it is possible that the truncated N terminus results from a sequence artifact (the homology extends further to the N terminus in another reading frame). Several other so-far-undefined EST clusters were found that have homology to PAGE as well as to GAGE. These clusters do not have the striking similarities that the other GAGE family members have to each other, but they are also not significantly more similar to PAGE than to GAGE. Representatives of some of these cDNA clusters are the ESTs yd88e11 (fetal liver/spleen), yw86a06 (placenta), and yi21h01 (placenta).

Relation of the sequences of GAGE, PAGE, and other so-far-uncharacterized EST clusters. The GCG program pileup was used to compare the multiple protein sequences of the GAGE and PAGE protein family. The dendrogram shows that PAGE proteins are a separate group of proteins that are less related to GAGE proteins.

Hybridization analysis of PAGE-1 expression. (A) A multiple tissue dot blot (Left) and Northern blots (Center and Right) were probed with a 140-bp ³²P-labeled PAGE-1 probe under very stringent hybridization conditions (50% formamide, 55°C). Specific PAGE-1 signals were observed in prostate, testis, placenta, and uterus, but not in other tissues (Table 1 legend lists the analyzed tissues). Because the hybridization probe had some similarity with another PAGE-1-like EST cluster that is expressed in testis (PAGE represented by the EST zv62h08, Table 2), we additionally used a probe with minimal homology to zv62h08 to confirm that the signal in testis corresponds to the expression of the authentic PAGE-1. (B) Blots containing 20 μg per lane total RNA from normal or malignant ovary (Right), fallopian tube (Center), and uterus (Left) were hybridized under stringent conditions. PAGE-1 is expressed in fallopian tube, uterus, and uterine cancer, but not in ovary and ovarian cancer.

RT-PCR analysis of PAGE-1 expression. Ethidium bromide-stained 2.5% agarose gel; PAGE-1 cDNA was amplified with 5′- and 3′-end-specific PAGE primers (40 cycles 94°C, 58°C, 72°C, 1 min each).