The identification and partial characterization of a human gene designated as AHNAK that encodes an unusually large protein are presented, and preliminary evidence indicates that the protein resides ...predominantly within the nucleus, but no function is discerned.
We have used cDNA subtractive cloning to identify a group of human genes that are expressed in diverse differentiated derivatives of neural crest origin but not in neuroblastoma cell lines. One of ...these genes was identified as CD44, which encodes an integral membrane glycoprotein that serves as the principal receptor for hyaluronate and participates in specific cell-cell and cell-extracellular matrix interactions. The repression of CD44 expression in neuroblastoma cell lines might be relevant to their high metastatic potential. We have cloned full-length cDNAs corresponding to CD44 transcripts and identified a novel splice variant of CD44 lacking 31 amino acids of the extracellular domain. As a first step toward analysis of CD44 downregulation in neuroblastoma cells, we have mapped the CD44 RNA initiation site and analyzed the structure of the upstream regulatory region. We constructed a series of plasmids containing different amounts of CD44 upstream regulatory region linked to the bacterial chloramphenicol acetyltransferase gene and then analyzed their ability to promote transcription in neuroblastoma and melanoma cells. We found that a DNA segment including about 150 bp of the CD44 upstream region and the 5′ end of the gene itself was sufficient to induce substantial transcription of the chloramphenicol acetyltransferase gene in both neuroblastoma and melanoma cells. Several upstream cis-acting elements contribute to the downregulation of CD44 in neuroblastoma cells, the most prominent being a 120-bp DNA fragment located 450 bp upstream to the RNA initiation site. Our data suggest that multiple factors might be involved in downregulation of CD44 in neuroblastoma cells.
Two mutation detection methods based on the cleavage of mismatched heteroduplexes were compared and evaluated. These techniques, chemical cleavage of mismatch (CCM) and enzyme mismatch cleavage ...(EMC), have the advantages over other available methods of being able to detect and localize mutations in relatively large fragments of DNA (> or = 1 kb). We have constructed clones that enable us to create heteroduplexes of 500 bp, 1 kb, and 1.5 kb and have assessed each of the methods over a range of criteria. Both were able to detect and localize all four types of single-base mismatch and insertion/deletions of 1-5 bp. Whereas EMC was efficient at detection of insertion/deletions in a broad size range of fragments and has the advantage over CCM of using no hazardous chemicals, in our hands it has not been sufficiently robust that we felt confident to consider it for diagnostic use in its present form. CCM using hydroxylamine was efficient over the entire range of fragment sizes tested and using potassium permanganate with tetraethylammonium chloride was efficient up to 1 kb.
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