ES cells express a SHIP mRNA species different from that expressed in mature hematopoietic cells.

(A) RT-PCR analysis of SHIP expression in ES cells and lineage-committed hematopoietic cell lines. All primers used in the RT-PCR assays were designed to amplify regions of SHIP mRNA encoded by separate exons; hence, the expected amplification products must arise from amplification of cDNA, not from contaminating genomic DNA. The following cell types were analyzed: B-lymphocyte cell lines 70Z/3 and WEHI-231, hepatoma cell line Hepa 1-6 (negative control), and ES cell lines TL1 and E10. Amplification of β-actin was included as a control for the RNA isolation and cDNA synthesis steps. PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. (B) RACE cloning of s-SHIP mRNA. PCR products representing the 5′ or 3′ termini of the s-SHIP mRNA expressed in ES cells were generated by 5′ and 3′ RACE, respectively. For 3′ RACE cloning of SHIP mRNA species in 70Z/3 and ES cells, the primers SHIP2766 (sense; lane 1) or SHIP1970 (sense; lane 2) were used. (C) Schematic depiction of the structure of the s-SHIP and SHIP cDNAs. Nucleotide numbering refers to GenBank accession numbers AF184912 and U52044, respectively. Functional domains and sequence motifs encoded in the s-SHIP and SHIP mRNAs ares-SHIP Region (SSR), Src-homology 2 domain (SH2), proline-rich motifs (PR), and NPXY and YIGM motifs. The internal Δ183 nucleotide deletion is illustrated. Initial methionine (M) and terminal glutamine (Q) amino acids for each mRNA are indicated, yielding proteins of 928 aa (s-SHIP) and 1191 aa (SHIP). Relative location and orientation of the primers used in the RT-PCR analysis or RACE cloning are indicated below the SHIP cDNA. Numbers below the primers (arrows) represent the 5′ nucleotide in the primer based on its position in the SHIP cDNA sequence.