Cloning of vertebrate TACC cDNAs
The rabbit TACC3 was cloned by rt-PCR using the "TACC4" specific primer T4RACE2 [9] (5'-cccgaactgctccaggtaatcgatctc-3') and a consensus primer, T3con2, designed to the region encompassing the vertebrate TACC3 initiator methionine (5'-tatgagtctgcaggtcttaaacgac-3'). For cloning the mouse Tacc1X cDNA, the primers used were based upon the genomic sequence reported, and the sequence of the IMAGE cDNA clone 4933429K08: T1XF (5'-ccatgttcagtcattggcaggtc-3'), T1XF2 (5'-ctgcagaaccaacagttcaag-3'), T1XR1 (5'-agatctgtgacatcacagctc-3'), T1XR2 (5'-ctcgagtcagttagtcttatccagctt-3'), BB617F (5'-accaccaacttgagtacctg-3') and BB617R (5'-gtatcttgaactgttggttctg-3'). For analysis of TACC1 splice variants, the forward primers used were located in exon 1b: EF (5'-gagagatgcgaaatcagcg-3'), Exon 1d: X3F (5'-agtcaaagaaggcatctgcag-3'), Exon 1a: 1DF (5'-ccaagttctgcgccatggg-3'). The reverse primers used were: Exon 1: X1R (5'-ggatttggtctcggcttgcgaatc-3'), Exon 2: BX647R (5'-cttgtgattcttggcttttgg-3'), Exon 3: 6SPR (5'-gtcatcgcctcgtcctggagggc-3'), Exon 4a: 1DR (5'-aatttcacttgttcagtagtc-3'), Exon 5: 128R (5'-cctgcttctgaggatgaaaacgc-3'). Rabbit brain poly A+ mRNA, mouse testis and human brain total RNA were obtained from BD Bioscience Clontech (Palo Alto, CA, U.S.A.). Reverse transcription and PCR was performed as previously described, using either 1 μg of total RNA or 50 ng of poly A+ mRNA as template for first strand cDNA synthesis. PCR products were cloned into pCR2.1 (Invitrogen, Carlsbad CA, U.S.A.) and transformed into InvαF' competent cells. Plasmid inserts were sequenced by the Roswell Park Cancer Institute Biopolymer Core Facility.