Studies on the Turtle Tumor Susceptibility Gene TSG101: Full-Length cDNA Sequence, Genomic Structural Analysis, and Role in Green Turtle Fibropapilloma

Abstract

The tumor susceptibility gene TSG101 is a recently discovered gene whose functional knockout in mouse fibroblasts leads to transformation and tumor formation in nude mice. Human and mouse TSG101 cDNAs are 86% and 94% similar at the nucleotide and deduced amino acid levels, respectively. The highly conserved protein sequences suggest that the mouse and human TSG101 are true gene homologs that share fundamental biological functions. Here, we report that the turtle TSG101 full-length cDNA sequence contained a 1,176-base-pair open translational reading frame predicted to encode a 392-amino-acid protein. Alignment of TSG101 sequences showed that the turtle cDNA sequence was 82.3% and 84.4% similar to mouse and human TSG101, respectively, at the nucleotide level and 89.3% and 91.9% similar to mouse and human TSG101 proteins, respectively. A coiled-coil domain and a proline-rich region typical of the activation domain of transcription factors were highly conserved among the turtle, mouse, and human TSG101. The leucine zipper motifs in the coiled-coil domains of turtle, mouse, and human TSG101 proteins were identical. Expression of TSG101 was observed in all turtle organs examined. The role of TSG101 in green turtle fibropapilloma (GTFP) was investigated by performing reverse transcriptase-polymerase chain reaction (RT-PCR) on RNA derived from various turtle tumor tissues and tumor cell lines. No transcript abnormalities of turtle TSG101 were found in all examined GTFP samples (10 GTFP tumor tissues and 2 GTFP tumor cell lines) from RT-PCR products. Future study will analyze the difference in turtle TSG101 expressions between GTFP and the corresponding normal tissue. In mammalian systems, TSG101 productions outside of a narrow range, either overexpression or deficiency, can lead to abnormal cell growth. It needs to be clarified whether turtle TSG101 in GTFP is up or down regulated.

Figure 1

Full-length sequence of the turtle tumor susceptibility gene TSG101 cDNA. The sites of the translation initiation and the translation stop code are shown in bold type. The primers for nested polymerase chain reaction (PCR) and the specific probe are underlined.

Figure 2

Protein sequences and features of turtle, mouse, and human tumor susceptibility gene TSG101. (A) Alignment of turtle, mouse, and human TSG101 protein sequences (labeled T-, M-, and H-TSG 101, respectively). Amino acid sequences of turtle, mouse, and human TSG101 proteins were predicted from TSG101 open reading frames determined by cDNA sequence analysis. The positions matched to turtle TSG101 are indicated by dots, and gaps between turtle and human or turtle and mouse TSG101 are indicated by dashes. The predicted coiled-coil domain is italicized, and the amino acid positions defining a leucine zipper motif within the coiled-coil domain are boxed. Amino-terminal to the coiled-coil domain is a proline-rich domain shown by the amino acid designation for proline (P) underlined. (B) Sequence alignment of TSG101 and cc2. Amino acid positions defining the leucine zipper motif are boxed. Sites of apparent sequence difference are indicated in bold type.

Figure 3

Nested PCR amplification of introns 8 and 9 of the turtle tumor susceptibility gene TSG101. The analysis was performed on turtle genomic DNA with exon-specific primers that encompassed individual introns. The gel lanes are as follows: lanes 1–3, intron 9 from turtles 1–3; lanes 4–6, intron 8 from turtles 1–3; and M, 1-kilobase ladder (Life Technologies). Two sets of primers for introns 8 (In8) and 9 (In9) were as follows: In8F1, 5′-GAAAGAGGAAATGGATCGTGCACAAGC-3′, and In8R1, 5′-GGATCTGTTTGTAAAGTGGTGCTGTTGG-3′; In8F2, 5′-CTTGGGAGAAGCATTGAGACGTGGGG-3′, and In8R2, 5-GAAGTTCAATGTTCTTATCGACTTCA-3′; In9F1, 5′-CCAACAGCACCACTTTACAAACAGATCC-3′, and In9R1, 5′-CTTCTGCATTAGTGCTCTCAGCTGG-3′; and In9F2, 5′-CTTGGGAGAAGC-ATTGAGACGTGGGG-3′, and In9R2, 5′-TGCTTGCGCGACAGAAGACGTACAT-3′. Nested PCR reactions were performed in a 50-μL solution containing 1 μg of genomic DNA, 200 ng of each primer, 0.2 mM dNTP, and 2.5 units of pfu Turbo DNA polymerase and 1× cloned pfu DNA polymerase reaction buffer (Stratagene). PCR amplifications were performed in a PE9700 thermal cycler at 95°C for 2 min, followed by 35 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 3 min. The inner PCR products were resolved in a 1.2% agarose gel and stained with ethidium bromide. The PCR products were cloned in vector pCR-Blunt (Invitrogen) and sequenced in both directions; bp = base pairs.

Figure 4

Turtle tumor susceptibility gene TSG101 mRNA expression in various organs. Ten microliters of reverse transcriptase (RT)-PCR products from each sample were separated by 1.2% agarose gel electrophoresis and visualized by ethidium bromide staining. The gel lanes are as follows: M, 100-bp ladder (Life Technologies); lanes 1–9, normal tissues of skin, eye, heart, kidney, spleen, urinary bladder, lung, liver, and tongue, respectively; and lane 10, control for reverse transcriptase dependence. An equal volume of RNA was used for cDNA synthesis without reverse transcriptase, and the resultant cDNA was used for the RT-PCR control.

Figure 5

Detection of turtle tumor susceptibility gene TSG101 transcripts in green turtle fibropapilloma (GTFP) tumor tissues and tumor cell lines. Ten microliters of RT-PCR products were separated by 1.2% agarose gel electrophoresis, visualized by ethidium bromide staining, and then transferred to nylon membranes. DNA was fixed by ultraviolet cross-linking and hybridized with a digoxigenin–dideoxyuridine 5′-triphosphate-labeled turtle TSG101 gene-specific probe. (A) PCR amplification of the full-length turtle TSG101 open reading frame. The gel lanes are as follows: M, 100-bp ladder (Life Technologies); lanes 1–3, skin, tongue, and lung tumors, respectively, from turtle 1 with fibropapilloma; lanes 4–6, skin, eye, and kidney tumors, respectively, from turtle 2 with fibropapilloma; lanes 7–9, skin, eye, and tongue tumors, respectively, from turtle 3 with fibropapilloma; lanes 10 and 11, cell lines from heart and lung tumors, respectively, of a turtle with GTFP; and lane 12, control for reverse transcriptase dependence. An equal volume of RNA was used for cDNA synthesis without reverse transcriptase, and the resultant cDNA was used for the RT-PCR control. (B) Southern blot analysis of the full-length turtle TSG101 open reading frame from RT-PCR results of A.