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. Author manuscript; available in PMC: 2022 Dec 16.
Published in final edited form as: J Aquat Anim Health. 2000 Dec;12(4):274–282. doi: 10.1577/1548-8667(2000)012<0274:sottts>2.0.co;2

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

Qigui Yu 1,*, Yuanan Lu 1, Vivek R Nerurkar 1, Richard Yanagihara 1
PMCID: PMC9757134  NIHMSID: NIHMS757044  PMID: 36530584

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.

The tumor susceptibility gene TSG101 is a recently discovered gene whose functional knockout in mouse fibroblasts leads to transformation and the ability to form tumors in nude mice (Li and Cohen 1996). Overexpression of TSG101 antisense RNA causes transformation of naïve 3T3 cells and elevation of stathmin mRNA (Li and Cohen 1996). Sequence analysis of mouse TSG101 cDNA indicates that the gene encodes a 43-kilodalton (kDa) protein containing a proline-rich domain and DNA-binding motifs characteristic of transcription factors (Li et al. 1997). Additionally, the TSG101 protein encodes a coiled-coil domain that is suggested to bind to oncoprotein 18 (Marklund et al. 1993), a cytoplasmic phosphoprotein elevated in a variety of malignancies and implicated in the regulation and relay of diverse signals associated with cell growth and differentiation (Hanash et al. 1988; Sobel 1991; Brattsand et al. 1993; Roos et al. 1993). The human homolog TSG101 has been mapped to chromosome 11, bands 15.1–15.2, a region proposed to contain tumor suppressor genes. The TSG101 cDNAs in mouse and human are 86% identical at the nucleotide level and 94% identical at the amino acid level as predicted from cDNAs. Highly conserved protein sequences suggest that the mouse and human TSG101 genes are true gene homologs and that they must have fundamental biological functions in common. The human TSG101 gene shows aberrant splicing in human breast cancer. The relaxation of RNA-splicing fidelity may contribute generally to abnormal gene expression in cancer and appears to be a novel oncodevelopmental marker for cancer.

Green turtle fibropapilloma (GTFP), a neoplastic disease characterized by epithelial fibropapillomas and internal fibromas, was first described in 1938 in green turtles Chelonia mydas from the Florida Keys (Lucke 1938). Since then, GTFP has been reported in green turtles worldwide with increasing frequency (Williams et al. 1994). Tumors are found on the skin, eyes, oral cavity, and carapace, as well as in visceral organs (Jacobson et al. 1989; Norton et al. 1990; Harshbarger 1991). The etiology of GTFP is unknown. Agents known to cause similar proliferative cutaneous lesions in other species, including chemical carcinogens, ultraviolet light, oncogenic viruses, metazoan parasites, and genetic factors, have been proposed as possible causes of GTFP (Sundberg 1991; Herbst and Klein 1995). The increased prevalence of GTFP makes the identification of the etiologic agent of paramount importance for the management of this disease and long-term survival of this endangered species, which is protected under provisions of the U.S. Endangered Species Act. Considerable circumstantial evidence supports a viral etiology of GTFP (Jacobson et al. 1991; Herbst et al. 1995, 1996), and a novel alpha-herpesvirus (green turtle herpesvirus [GTHV]) has recently been identified and implicated (Quackenbush et al. 1998). Sequences of GTHV have been detected by polymerase chain reaction (PCR) in tumor tissues of affected green turtles (Quackenbush et al. 1998; Lackovich et al. 1999). Unfortunately, no virus has been successfully isolated in cell cultures. On the other hand, genetic factors have been thought to play a role in the pathogenesis of GTFP. However, it is not known whether the green turtle has oncogenes similar to those in mammalian systems and higher warm-blooded vertebrates and whether these oncogenes are related to GTFP. In this study, we cloned, sequenced, and analyzed the homologs of the human and mouse tumor susceptibility gene TSG101 from turtle tissues. The role of TSG101 in the pathogenesis of green turtle fibropapillomatosis was investigated by performing reverse transcriptase (RT)-PCR on RNA derived from various turtle tumor tissues and tumor cell lines. No transcript abnormalities of turtle TSG101 were found in GTFP from RT-PCR products. Study of the difference in turtle TSG101 expressions between GTFP and the corresponding normal tissue is under way.

Methods

Tissues and cell lines

Green turtles with or without GTFP, captured in waters surrounding the Hawaiian Islands, were euthanatized by members of the U.S. Fish and Wildlife Service. Nontumor and tumor tissues, including sections of skin, eye, heart, kidney, spleen, urinary bladder, lung, liver, and tongue, were collected aseptically at autopsy. Cell lines of heart and lung tumors were established from a green turtle with GTFP (Lu et al. 1999).

Isolation of DNA and RNA from nontumor and tumor tissues and cell lines

For DNA isolation, tissues and cell lines were digested in an extraction buffer (10 mM tris-HCl [pH 8.0], 0.1 M EDTA [pH 8.0], 20 μg pancreatic RNase/mL, 0.5% sodium dodecyl sulfate [SDS], and 0.2 mg proteinase K/mL) at 50°C overnight. The digestion solution was extracted once with phenol (tris-HCl saturated, pH 8.0), then with phenol: chloroform: isopropanol (25:24:1), and finally with chloroform: isopropanol (24:1). The DNA was precipitated by adding 2.5 volumes of 100% ethanol and 0.1% 3 M sodium acetate (pH 5.2). Total RNA was isolated from 106 cells of each cell line or 200 mg of each tissue (dissected into pieces) by the use of the RNAzol B Isolation Kit (Tel-Test, Inc.) and treated with RNase-free DNase (Invitrogen Corp.) to remove any residual DNA contamination. Poly-A+ RNA was purified from total RNA by the use of the Oligotex mRNA Kit (QIAGEN, Germany) according to the manufacturer’s instructions. Total RNA and poly-A+ RNA were stored at −80°C until used.

RT-PCR and cDNA sequencing

Total RNA (2 μg) or poly-A+ RNA (100 ng) was mixed with 500 ng of oligo(dT)18 or the outer antisense primer in 12 μL of diethyl pyrocarbonate water and denatured at 70°C for 10 min; the reverse transcription reaction was performed in a 20-μL solution containing 1× buffer (50 mM tris-HCl [pH 8.3], 75 mM KCl, and 3 mM MgCl2), 10 mM dithiothreitol, 500 nM deoxynucleotide triphosphate (dNTP) mix, 1 unit RNase inhibitor/μL, and 200 units of SuperScript II RNase H− reverse transcriptase (Life Technologies). The reaction mixture was incubated at 42°C for 1 h, and cDNA was stored at −80°C. Primers corresponding to the noncoding regions of human and mouse TSG101 cDNA sequences were used to amplify the turtle full-length TSG101 cDNA. Nested RT-PCR was performed in 50-μL mixtures containing 5 μL of cDNA, 10 mM tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% (weight: volume) gelatin, 200 M dNTP, 2 units of Taq (Perkin-Elmer, Branchburg, New Jersey), and 200 ng of each primer (outer primers: 5′-CGGTCTAGGGCAGCCTATCAGC-3′ and 5′-AACTGCAATAACTTATTGTGGG-3′; inner primers: 5′-CTCTGCCTGTGGGGAAGGAGGT-3′ and 5′-AGCTCAACCTCCAGCTGGTATC-3′). The PCR amplifications were performed in a PE9700 DNA 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 2 min. Controls for reverse transcriptase dependence and DNA contamination were included. The products obtained by PCR were size fractionated on 1.2% agarose gels and visualized by ethidium bromide staining and then cloned in vector pCR2.1 (Invitrogen) and sequenced in both directions by the use of the BigDye Kit (Perkin-Elmer) in an automated DNA sequencer (model 377; Applied Biosystems, Inc.).

Southern blot analysis

Turtle TSG101 RT-PCR products, amplified from cDNA of various tissues and cell lines, were separated by agarose gel electrophoresis and then transferred to nylon membranes (Boehringer Mannheim). The DNA, fixed by ultraviolet cross- linking at 254 nm for 3 min (Stratalinker; Stratagene), was hybridized with a digoxigen-in–dideoxyuridine 5′-triphosphate-labeled specific probe corresponding to turtle TSG101 exon 9 (5′-CCAACAGCACCACTTTACAAACAG-3′) at 56°C for 8 h. Membranes were then washed twice for 5 min each at 56°C with 2× standard sodium citrate (SSC) (0.3 M NaCl and 30 mM Na-citrate, pH 7.0) containing 0.1% (weight: volume) SDS and twice for 5 min each at 56°C with 0.1× SSC containing 0.1% (weight: volume) SDS. Detection of the digoxigenin-labeled nucleic acids with disodium 3-(4-methoxyspiro{1,2-dioxetane-3, 2′-(5′-chloro) tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (Boehringer Mannheim) was performed according to the manufacturer’s instructions. Exposures to X-ray film were used to detect the hybridization signal.

Results

Cloning and Characterization of Turtle TSG101 cDNA

To search for the etiology of GTFP, we performed cDNA subtractive hybridization by use of the CLONTECH PCR-Select cDNA Subtraction kit (CLONTECH Laboratories, Inc.) according to the manufacturer’s instructions. From a green turtle with GTFP, tongue tumor tissue and normal-appearing tongue tissue were collected and used for RNA isolations. Tester cDNA synthesized from poly-A+ RNA isolated from the tumor tongue tissue was subtracted against driver cDNA synthesized from poly-A+ RNA isolated from the normal-appearing tongue tissue. A 450-base pair (bp) fragment (z1898) was cloned from the products of the cDNA subtractive hybridization. Sequence analysis of z1898 showed 84.3% and 86% identity at the nucleotide level to murine and human TSG101, respectively. At the amino acid level, the sequence similarity exceeded 98%. Primers corresponding to the sequences of human and mouse TSG101 cDNA were used to amplify full-length turtle TSG101 cDNA by nested RT-PCR, employing as template mRNA purified from total RNA from turtle tongue tissue by the Oligotex mRNA Kit (QIAGEN).

A 1,245-bp fragment was amplified and cloned from turtle cDNA. The sequence of this 1,245-bp fragment was 82.3% and 84.4% identical to mouse and human TSG101, respectively, at the nucleotide level. The 1,245-bp sequence contained a 1,176-bp open reading frame, with a coding capacity for a 392-amino acid protein (Figure 1). The predicted turtle TSG101 protein was 89.3% and 91.9% identical, respectively, with mouse and human TSG101 proteins. From the amino terminus of TSG101 proteins, only three amino acids were mismatched between the turtle and mouse, and two amino acids were mismatched between the turtle and human within 186 amino acids (Figure 2A). A coiled-coil domain and a proline-rich region were conserved among turtle, mouse, and human TSG101; only one amino acid mismatch was between turtle and human and between mouse and human in coiled-coil domains, and two amino acid mismatches were between turtle and mouse TSG101 in the coiled-coil domains. The leucine zipper motifs in the coiled-coil domains of turtle, mouse, and human TSG101 proteins were identical to each other (Figure 2B). Other features conserved among turtle, mouse, and human TSG101 included putative protein kinase C phosphorylation sites (amino acids [aa] 21, 48, 96, 99, 227, 237, and 369), five potential casein kinase II phosphorylation sites (aa 48, 222, 261, 277, and 301), and two potential N-glycosylation sites (aa 161 and 308) (Bairoch and Bucher 1994).

Figure 1.

Figure 1

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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.

Figure 2

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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.

Analysis of Partial Genomic Structure of Turtle TSG101

Initially, human TSG101 gene architecture was considered to contain six exons and five introns by restriction endonuclease analysis (Li et al. 1997). But the result of PCR amplification of human genomic DNA determined with primers corresponding to the human coding sequence, which flank the anticipated intron sites, indicated that there were four additional introns in the sequence considered to be the first exon (Wagner et al. 1998). To compare the location and size of the turtle TSG101 gene with the human and mouse homologs, we designed primers corresponding to the turtle coding sequence, which flank the anticipated sites of introns 8 and 9. Turtle introns 8 (3 kilobases [kb]) and 9 (2.6 kb) were much larger than the predicted human introns 8 (2.0 kb) and 9 (1.1 kb) (Li et al. 1997) and mouse introns 8 (1.2 kb) and 9 (1.2 kb) (Figure 3). Sequence analysis showed that turtle intron 8 was located between nucleotides 892 and 893 and that intron 9 was located between nucleotides 1,132 and 1,133. Alignment of intron–exon boundaries among turtle, human, and mouse indicated that the sequences of the boundaries were conserved, but with two to three nucleotides different (Table 1).

Figure 3.

Figure 3

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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.

Table 1.

Comparison of the nucleotide sequence of intron–exon boundaries of turtle, human, and mouse TSG101 genes; ND = not determined.

5′ donor sequence
 Intron 8
size (kb)a 		5′ donor sequence
 Intron 9
size (kb)
Exon 8 Intron 8 Exon 9 Intron 9
Turtle CCACGAAGTG gtacctgact 3.0 CTTTCTAAAG aggtttcgc 2.6
Human TCAAGAAGTG gtaagtgact 2.3 CTTCCTGAAG gtatttcttc 1.2
Mouse TCAAGAAGTG ND 2.3 GTTCCTGAAA ND 1.2
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a

Kilobases.

Expression of the Turtle TSG101 Gene in Various Organs

Expression of TSG101 was analyzed in several turtle cell lines and organs, including skin, eye, heart, kidney, spleen, urinary bladder, lung, liver, and tongue, by RT-PCR (Figure 4). The expression of TSG101 was detected in all organs examined. The RT-PCR data support the ubiquitous expression of TSG101 in all turtle organs and as found previously in various human and mouse tissues.

Figure 4.

Figure 4

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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.

Absence of TSG101 Transcript Abnormalities in GTFP

We investigated the role of TSG101 in GTFP by performing RT-PCR on RNA derived from various turtle tissues and cell lines. The PCR primers were designed from the 5′- and 3′-untranslated regions of turtle TSG101 to encompass the entire coding region of the gene. Representative results depicted in Figure 5 show that in all cases a major transcript of 1.2 kb and no smaller aberrant bands were seen.

Figure 5.

Figure 5

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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.

Discussion

The mouse and human TSG101 full-length cDNAs, which are 86% identical at the nucleotide level, contain a 1,173- and 1,170-bp open reading frame predicted to encode a 391- and 390-amino acid protein, respectively, of 43 kDa in size showing 94% sequence similarity. By comparison, the turtle TSG101 full-length cDNA sequence contained a 1,176-bp open reading frame predicted to encode a 392-amino acid protein, approximately 90% homologous to the mouse and human homologs. A coiled-coil domain and a proline-rich region typical of the activation domain of transcription factors (Mitchell and Tjian 1989) were highly conserved among turtle, mouse, and human TSG101. The leucine zipper motifs (Landschulz et al. 1988; O’Shea et al. 1989a, 1989b) in the coiled-coil domains of turtle, mouse, and human TSG101 proteins were identical to each other. Very recently, it is reported that intracellular TSG101 protein is in fact maintained within a narrow range in cultured mammalian cell populations by a post-translational mechanism that modulates TSG101 protein degradation and prevents its accumulation in cells overexpressing TSG101 mRNA. The regulation of the steady-state level of intracellular TSG101 protein is mediated by an evolutionarily conserved sequence (20 amino acids in length from the end of the carboxyl terminus) located near its carboxyl terminus (termed the SB4; Feng et al. 2000). It is very interesting that the carboxyl terminus, including SB4 and its flanking amino acid sequence, of turtle TSG101 mRNA is identical to that in human and mouse (Figure 2A). We speculated that the homologs of SB4 in turtle TSG101 might play a key role in regulation of the steady-state level of intracellular TSG101 protein.

Taken together, mRNA and amino acid sequences of the mouse, human, and turtle TSG101 protein were highly conserved, suggesting that the TSG101 genes may have fundamental biological functions in common.

The mouse TSG101 gene consists of 9 introns and 10 exons and spans about 33.6 kb (Wagner et al. 1998). By contrast, the human TSG101 gene cloned from the PAC library was initially estimated to be much smaller in size and to contain six exons (Li et al. 1997). However, four additional introns in the first exon of the human TSG101 gene were recently found (Wagner et al. 1998). In our study, introns 8 and 9 of the turtle TSG101 were much larger than those in the human and mouse homologs.

The TSG101 gene was initially identified in mouse National Institutes of Health 3T3 fibroblasts by a novel gene-discovery approach (random homozygous knock out) that enables regulated functional inactivation of multiple copies of previously unknown genes and selection for cells that show a phenotype resulting from such inactivation (Li et al. 1997). The functional inactivation of this gene leads to cell transformation in vitro and to metastasizing tumors in vivo when transformed mouse fibroblasts are transplanted into nude mice (Li and Cohen 1996). The human TSG 101 gene has been mapped to a specific region on chromosome 11, p15.1–p15.2, which is associated with a loss of heterozygosity in different types of malignancies such as breast cancer and Wilms’ tumor (Reeve et al. 1989). However, rearrangements and somatic mutations within the TSG101 gene are rare events in human breast cancers (Lee and Feinberg 1997; Steiner et al. 1997; Trink et al. 1998). It was proposed that shorter transcripts are caused by abnormal splicing events recognizing cryptic splicing donor and acceptor sites within exons (Gayther et al. 1997; Lee and Feinberg 1997). We amplified the entire coding region of the turtle TSG101 gene from 10 GTFP tumor tissues and 2 GTFP tumor cell lines. No TSG101 transcript abnormalities were found in GTFP. However, the difference in TSG101 expressions between GTFP and the corresponding normal tissue needs to be examined because mammalian TSG101 products outside of a narrow range, either overexpression or deficiency, can lead to abnormal cell growth (Feng et al. 2000).

The cause of GTFP has not been determined, and it is not clear whether overexpression or deficiency of turtle TSG101 plays a role in the pathogenesis of GTFP. However, considerable circumstantial evidence supports a viral etiology of GTFP (Jacobson et al. 1991; Herbst et al. 1995, 1996). GTFP has been induced experimentally in green turtles after intramuscular inoculation with cell-free, tumor-tissue homogenates (Herbst et al. 1995). Moreover, herpesvirus particles have been visualized in experimentally induced tumors by transmission electron microscopy (Herbst 1994; Aguirre and Sprraker 1996), and a novel alpha-herpesvirus (green turtle herpes virus, GTHV) has recently been identified and implicated (Quackenbush et al. 1998). The GTHV sequences have been detected by PCR in tumor tissues of affected turtles (Quackenbush et al. 1998; Lackovich et al. 1999). The widespread distribution of GTHV in internal and external tumors and its absence in nontumored animals strongly suggest an etiologic association between GTHV and GTFP (Lu et al. 2000). Unfortunately, no virus has been successfully isolated in cell cultures.

Acknowledgments

We thank Thierry M. Work of the National Wild-life Health Center, George H. Balazs of the National Marine Fisheries Services, and A. Aguirre of the Wildlife Clinic, Tufts University School of Veterinary Medicine, for their assistance in collecting turtle tissue specimens. This research was supported in part by a contract from the National Marine Fisheries Service Southwest Fisheries Science Center, Honolulu Laboratory, and by grants from the Hawaii Community Foundation (435073) and the U.S. Public Health Service (G12RR/AI-03061) from the Research Centers in Minority Institutions Program of the National Center for Research Resources, National Institutes of Health.

Footnotes

The sequence described in this paper have been deposited with the GENBANK database and has the accession number AF279276.

References

  1. Aguirre AA, Sprraker TR. Microscopic and ultrastructural evidence of herpesvirus-like virus in Hawaiian green turtles (Chelonia mydas) with fibropapillomatosis. NOAA; Honolulu: 1996. Southwest Fisheries Center Administrative Report H-96-06C. [Google Scholar]
  2. Bairoch A, Bucher P. Prosite: recent developments. Nucleic Acids Research. 1994;22:3583–3589. [PMC free article] [PubMed] [Google Scholar]
  3. Brattsand G, Roos G, Marklund U, Ueda H, Landberg G, Nanberg E, Sideras P, Gullberg M. Quantitative analysis of the expression and regulation of an activation-regulated phosphoprotein (oncoprotein 18) in normal and neoplastic cells. Leukemia. 1993;7:569–579. [PubMed] [Google Scholar]
  4. Feng GH, Lih CJ, Cohen SN. TSG101 protein steady-state level is regulated posttranslationally by an evolutionarily conserved COOH-terminal sequence. Cancer Research. 2000;60:1736–1741. [PubMed] [Google Scholar]
  5. Gayther SA, Barski P, Batley SJ, Li L, de Foy KA, Cohen SN, Ponder BA, Caldas C. Aberrant splicing of the TSG101 and FHIT genes occurs frequently in multiple malignancies and in normal tissues and mimics alterations previously described in tumours. Oncogene. 1997;15:2119–2126. doi: 10.1038/sj.onc.1201591. [DOI] [PubMed] [Google Scholar]
  6. Hanash SM, Strahler JR, Kuick R, Chu EH, Nichols D. Identification of a polypeptide associated with the malignant phenotype in acute leukemia. Journal of Biological Chemistry. 1988;263:12813–12815. [PubMed] [Google Scholar]
  7. Harshbarger JC. Sea turtle fibropapilloma cases in the registry of tumors in lower animals: research plan for marine turtle fibropapilloma. 1991. pp. 63–70. NOAA Technical Memorandum NMFS-SWFSC-156. [Google Scholar]
  8. Herbst LH. Fibropapillomatosis of marine turtles. Annual Review Fish Diseases. 1994;4:389–425. [Google Scholar]
  9. Herbst LH, Jacobson ER, Moretti R, Brown T, Sundberg JP, Klein PA. Experimental transmission of green turtle fibropapillomatosis using cell-free tumor extracts. Diseases of Aquatic Organisms. 1995;22:1–12. [Google Scholar]
  10. Herbst LH, Klein PA. Green turtle fibropapillomatosis: challenges to assessing the role of environmental cofactors. Environmental Health Perspective. 1995;103(Supplement 4):27–30. doi: 10.1289/ehp.95103s427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Herbst LH, Moretti R, Brown T, Klein PA. Sensitivity of the transmissible green turtle fibropapillomatosis agent to chloroform and ultracentrifugation conditions. Diseases of Aquatic Organisms. 1996;25:225–228. [Google Scholar]
  12. Jacobson ER, Buergelt C, Williams B, Harris RK. Herpesvirus in cutaneous fibropapillomas of the green turtle Chelonia mydas. Diseases of Aquatic Organisms. 1991;12:1–6. [Google Scholar]
  13. Jacobson ER, Mansell JL, Sundberg JP, Hajjar L, Reichmann ME, Ehrhart LM, Walsh M, Murru F. Cutaneous fibropapillomas of green turtles (Chelonia mydas) Journal of Comparative Pathology. 1989;101:39–52. doi: 10.1016/0021-9975(89)90075-3. [DOI] [PubMed] [Google Scholar]
  14. Lackovich JK, Brown DR, Homer BL, Garber RL, Mader DR, Moretti RM, Patterson AD, Herbst LM, Oros J, Jacobson ER, Curry SS, Klein PA. Association of herpesvirus with fibropapillomatosis of the green turtle Chelonia mydas and the loggerhead turtle Caretta caretta in Florida. Diseases of Aquatic Organisms. 1999;37:89–97. doi: 10.3354/dao037089. [DOI] [PubMed] [Google Scholar]
  15. Landschulz WH, Johnson PF, McKnight SL. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988;240:1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  16. Lee MP, Feinberg AP. Aberrant splicing but not mutations of TSG101 in human breast cancer. Cancer Research. 1997;57:3131–3134. [PubMed] [Google Scholar]
  17. Li L, Li X, Francke U, Cohen SN. The TSG101 tumor susceptibility gene is located in chromosome 11 band p15 and is mutated in human breast cancer. Cell. 1997;88:143–154. doi: 10.1016/s0092-8674(00)81866-8. [DOI] [PubMed] [Google Scholar]
  18. Li L, Cohen SN. Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous knockout of allelic loci in mammalian cells. Cell. 1996;85:319–329. doi: 10.1016/s0092-8674(00)81111-3. [DOI] [PubMed] [Google Scholar]
  19. Lu Y, Nerurkar VR, Aguirre AA, Work TM, Balazs GH, Yanagihara R. Establishment and characterization of 13 cell lines from a green turtle (Chelonia mydas) with fibropapillomas. In Vitro. 1999;35:389–393. doi: 10.1007/s11626-999-0113-6. [DOI] [PubMed] [Google Scholar]
  20. Lu Y, Wang Y, Yu Q, Aguirre AA, Balazs GH, Nerurkar VR, Yanagihara R. Detection of herpesviral sequences in tissues of green turtles with fibropapilloma by polymerase chain reaction. Archives of Virology. 2000;145:1–9. doi: 10.1007/s007050070063. [DOI] [PubMed] [Google Scholar]
  21. Lucke B. Studies on tumors in cold-blooded vertebrates. Vol. 1937. Carnegie Institution of Washington, Annual Report of Tortugas Laboratory; 1938. pp. 92–94. [Google Scholar]
  22. Marklund U, Brattsand G, Shingler V, Gullberg M. Serine 25 of oncoprotein 18 is a major cytosolic target for the mitogen-activated protein kinase. Journal of Biological Chemistry. 1993;268:15039–15047. [PubMed] [Google Scholar]
  23. Mitchell PJ, Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989;245:371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  24. Norton TM, Jacobson ER, Sundberg JP. fibropapillomas and renal myxofibroma in a green turtle, Chelonia mydas. Journal of Wildlife Diseases. 1990;26:265–270. doi: 10.7589/0090-3558-26.2.265. [DOI] [PubMed] [Google Scholar]
  25. O’Shea EK, Rutkowski R, Kim PS. Evidence that the leucine zipper is a coiled coil. Science. 1989a;243:538–542. doi: 10.1126/science.2911757. [DOI] [PubMed] [Google Scholar]
  26. O’Shea EK, Rutkowski R, Stafford WF, III, Kim PS. Preferential heterodimer formation by isolated leucine zippers from fos and jun. Science. 1989b;245:646–648. doi: 10.1126/science.2503872. [DOI] [PubMed] [Google Scholar]
  27. Quackenbush SL, Work TM, Balazs GH, Casey RN, Rovnak J, Chaves A, DuToit L, Baines JD, Parrish CR, Bowser PR, Casey JM. Three closely related herpesviruses are associated with fibropapillomatosis in marine turtles. Virology. 1998;246:392–399. doi: 10.1006/viro.1998.9207. [DOI] [PubMed] [Google Scholar]
  28. Reeve AE, Sih SA, Raizis AM, Feinberg AP. Loss of allelic heterozygosity at a second locus on chromosome 11 in sporadic Wilms’ tumor cells. Molecular and Cellular Biology. 1989;9:1799–1803. doi: 10.1128/mcb.9.4.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roos G, Brattsand G, Landberg G, Marklund U, Gullberg M. Expression of oncoprotein 18 in human leukemias and lymphomas. Leukemia. 1993;7:1538–1546. [PubMed] [Google Scholar]
  30. Sobel A. Stathmin: a relay phosphoprotein for multiple signal transduction? Trends in Biochemical Sciences. 1991;16:301–305. doi: 10.1016/0968-0004(91)90123-d. [DOI] [PubMed] [Google Scholar]
  31. Steiner P, Barnes DM, Harris WH, Weinberg RA. Absence of rearrangements in the tumor susceptibility gene in human breast cancer. National Genetics. 1997;16:332–333. doi: 10.1038/ng0897-332. [DOI] [PubMed] [Google Scholar]
  32. Sundberg JP. Research plan for marine turtle fibropapilloma. 1991. pp. 75–76. NOAA Technical Memorandum NMFS-SWFSC-156. [Google Scholar]
  33. Williams EH, Bunkley-Williams L, Peters EC, Pinto Rodriquez B, Matos-Morales R, Mignucci-Giannnoni AA, Hall K, Rueda-Almonacid JV, Sybesma J, DeCalventi IB, Boulon RH. An epizootic of cutaneous fibropapillomas in green turtles Chelenia mydas of the Caribbean: part of a panzootic? Journal of Aquatic Animal Health. 1994;6:70–78. [Google Scholar]
  34. Trink B, Pai SI, Spunt SL, Raman V, Cairns P, Jen J, Gabrielson E, Sukumar S, Sidransky D. Absence of TSG101 transcript abnormalities in human cancers. Oncogene. 1998;16:2815–2818. doi: 10.1038/sj.onc.1201795. [DOI] [PubMed] [Google Scholar]
  35. Wagner KU, Dierisseau P, Rucker EB, III, Robinson GW, Hennighausen L. Genomic architecture and transcriptional activation of the mouse and human tumor susceptibility gene TSG101: common types of shorter transcripts are true alternative variants. Oncogene. 1998;17:2761–2770. doi: 10.1038/sj.onc.1202529. [DOI] [PubMed] [Google Scholar]

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