METHODS

Construction of prey library composed of long cDNAs (KIAA clones)

In this study, we focused on binding of the cytoplasmic domain of a large, transmembrane protein with proteins localized in the submembrane region. To minimize false positive identification of interactions with proteins believed not to be co-localized in vivo, the nuclear and extracellular proteins located in domains located outside of the cytoplasm (i.e., putative transcription factors and extracellular domains of the transmembrane protein) were removed from the prey library. This was achieved by analyzing the amino acid sequence of KIAA genes using Pfam searches and the SOSUI program. KIAA genes containing motifs for transcription factors ( e.g., Pfam ID, PF00010, PF00096, PF00170, PF00249, PF00628, PF00642, PF00643 and PF01530) and the predicted transmembrane region were omitted from the library used for this study. Plasmids totaling 1087 KIAA clones ( supplemental Table 1 summarizing these details is available on-line at http://www.genome.org) were pooled at almost equal ratios, sonicated and blunt-ends were produced using mung bean nuclease and T4 DNA polymerase (TAKARA).

DNA fragments ranging from 1 kbp to 2 kbp were isolated electrophoretically in agarose gels (low melting point temperature), then cloned between the DraI and EcoRV sites of the pENTR 1A vector (Invitrogen). A pool of cDNA plasmids was recovered from 3.5 x 106 primary transformants (ElectroMax DH10B cells, Invitrogen) and grown on agar plates containing 30 mg/ml of kanamycin. To construct the destination plasmid pACTGW-attR, plasmid attR-pSP73 (Ohara and Temple, 2001) was double-digested with HindIII/SacI, an attR cassette containing the ccdB and chloramphenicol-resistance genes was isolated and ligated into the SmaI-XhoI site of pACT2 (Clontech) with blunt-ends. A pool of cDNA plasmids was converted to expression clones via the Gateway LR reaction (Invitrogen) using pACTGW-attR. The reaction products were transformed into E. coli cells. Plasmids were recovered from transformants (7.3 x 106 ) on ampicillin-containing agar plates after growth for 14 h at 30¡ëC. The cDNA plasmids were transformed into yeast, Y187 (MATa). Transformants (1.6 x 107 ) were grown on SD (synthetic medium)/-Leu plates and pooled. Aliquots from the pooled yeast transformant were used for mating in two-hybrid screening.

Preparation of bait and mating-mediated yeast two-hybrid screening

To construct the destination plasmid pASGW-attR, plasmid attR-pSP73 was double-digested with HindIII/SacI, an attR cassette containing the ccdB and chloramphenicol-resistance genes was isolated and ligated into the EcoRI-SalI site of pAS2-1 (Clontech) with blunt-ends. Among the KIAA proteins, KIAA genes encoding transmembrane regions were selected using predictions from the SOSUI program. Using KIAA cDNA as a template, each cDNA fragment containing a cytoplasmic domain was amplified by PCR and oligomers GWF and GWR (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTG(X)24-3' and 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTA(X)24-3', respectively ((X)24 is the gene-specific sequence). Supplemental Table 2 describing these cDNAs is available on-line at http://www.genome.org. Following the BP reaction, the attB-PCR product was introduced into expression clones via the Gateway LR reaction using pASGW-attR and a donor plasmid (Invitrogen). These clones were introduced into E. coli DH5a, and the inserts of all bait plasmids were confirmed by DNA sequencing. The bait plasmid was transformed into yeast AH109 (MATa), and transformants were grown on SD/-Trp plates. Self-activation assays were performed on SD/-Ade/-His/-Trp plates. Two transformants per each bait exhibiting no background growth on -Ade/-His were pooled, cultured and mated with an aliquot of yeast prey containing the transformant library. Diploid cells were spread onto 9 cm x 13 cm 20-25 plates containing SD/-Ade/-His/-Leu/-Trp/15mM 3-AT (3-amino-1,2,4-triazole). After 7-10 days of growth at 30¡ëC, typically 30 colonies were re-plated onto SD/-Ade/-His/-Leu/-Trp/15mM 3-AT plates containing X-a-gal. Blue-colored colonies were selected and cultured in SD/-Leu medium. Each plasmid was purified from the yeast and used as a template for PCR with oligomers OADN and OADC (5'-TCG ATG ATG AAG ATA CCC CAC-3', 5'-AAG AAA TTG AGA TGG TGC ACG-3', respectively). DNA sequences of prey clones were determined to identify the interacting region of the KIAA protein. The resulting sequences were compared with the HUGE database (http://www.kazusa.or.jp/huge/) using Blast2.