Characterization of vtg-1 mRNA expression during ontogeny in Oreochromis mossambicus (PETERS).

The yolk-precursor lipoprotein, vitellogenin (VTG) has been widely recognized as a biomarker for the detection of estrogenic activity in water-borne chemical pollutants. We characterized the expression status of this important constituent of reproduction in the Mozambique tilapia (Oreochromis mossambicus), a tilapiine freshwater fish species indigenous to Southern Africa, and investigated its utility in detection of exposure to estrogen using a quantitative real-time polymerase chain reaction (QPCR) assay. We initially isolated a 3kb upstream promoter region of the vtg gene and identified putative binding sites for several regulatory factors including estrogen receptor (ESR). Evidence for the expression of several splice-site vtg mRNA variants was found in a number of tissue types. A quantitative real-time polymerase chain reaction (QPCR) assay was subsequently developed based upon a specific primer pair (OMV6/9) that selectively amplified the liver-enriched transcript. The level of this transcript in liver tissue was high in females and lower, but detectable, in males and was significantly increased in male fish following laboratory exposure to 17beta-estradiol (E(2)). This study further established that juvenile whole body homogenates (WBHs) contained extremely low levels of liver-specific vtg mRNA between 5 and 110 days post-fertilization (dpf) compared to adult male liver. Subsequent exposure of 20 dpf juveniles to E(2) showed a substantial increase in this transcript within hours, and when compared to classic male model under same conditions, the juveniles were remarkably more sensitive. We therefore conclude that the quantification, using QPCR methodology, of vtg mRNA expression in 20 dpf O. mossambicus juveniles has promise for assessing estrogenic EDC activity in aquatic sources.

Ontogenetic changes in photoreceptor opsin gene expression in coho salmon (Oncorhynchus kisutch, Walbaum).

Pacific salmonids start life in fresh water then migrate to the sea, after a metamorphic event called smoltification, later returning to their natal freshwater streams to spawn and die. To accommodate changes in visual environments throughout life history, salmon may adjust their spectral sensitivity. We investigated this possibility by examining ontogenetic and thyroid hormone (TH)-induced changes in visual pigments in coho salmon (Oncorhynchus kisutch, Walbaum). Using microspectrophotometry, we measured the spectral absorbance (quantified by lambda(max)) of rods, and middle and long wavelength-sensitive (MWS and LWS) cones in three age classes of coho, representing both freshwater and marine phases. The lambda(max) of MWS and LWS cones differed among freshwater (alevin and parr) and ocean (smolt) phases. The lambda(max) of rods, on the other hand, did not vary, which is evidence that vitamin A(1)/A(2) visual pigment chromophore ratios were similar among freshwater and ocean phases when sampled at the same time of year. Exogenous TH treatment long wavelength shifted the lambda(max) of rods, consistent with an increase in A(2). However, shifts in cones were greater than predicted for a change in chromophore ratio. Real-time quantitative RT-PCR demonstrated that at least two RH2 opsin subtypes were expressed in MWS cones, and these were differentially expressed among alevin, parr and TH-treated alevin groups. Combined with changes in A(1)/A(2) ratio, differential expression of opsin subtypes allows coho to alter the spectral absorbance of their MWS and LWS cones by as much as 60 and 90 nm, respectively. To our knowledge, this is the largest spectral shift reported in a vertebrate photoreceptor.

Detection of environmental endocrine-disruptor effects on gene expression in live Rana catesbeiana tadpoles using a tail fin biopsy technique.

We describe a novel method of rapidly assessing exposure of premetamorphic Rana catesbeiana tadpoles to endocrine-disrupting chemicals. Tail fin biopsy and reverse transcription-polymerase chain reaction analyses of the thyroid hormone (TH)-responsive gene, TH receptor beta (TRbeta), demonstrate for the first time that a known accelerant of TH-induced metamorphosis, acetochlor, can significantly enhance TH-induced TRbeta mRNA levels within 24 h at an environmentally relevant dose. Although we focus on laboratory exposures in this study, this method easily can be adapted for use in field studies.

A novel exon within the mdm2 gene modulates translation initiation in vitro and disrupts the p53-binding domain of mdm2 protein.

The mdm2 protein interacts with a number of proteins involved in cell growth control. Such interactions favour cell proliferation and may explain the oncogenic potential of mdm2 when over-expressed in cells. Interaction with the tumour suppressor p53 involves the N-terminus of mdm2 and targets p53 for rapid degradation by the ubiquitin pathway. We now describe a novel, highly conserved exon of mdm2 (exon alpha) which includes an in-frame UGA stop codon. Expression of exon alpha disrupts in vitro translation of the p53 binding domain of mdm2. We propose that exon alpha induces translation re-initiation at an internal AUG codon within the mdm2 alpha mRNA isoform. The putative mdm2 alpha protein lacks the N-terminus of mdm2 and shows little, if any, binding capacity for p53. Mdm2 alpha mRNA is expressed in a tissue-specific manner and is observed predominantly in testis and peripheral blood lymphocytes. We propose that mdm2 alpha expression may provide a mechanism for uncoupling mdm2-p53 interaction in certain cell types and/or under specific conditions of cell growth.

Identification of tumour-associated and germ line p53 mutations in canine mammary cancer.

Mutations of the tumour suppressor p53 gene are found in a number of spontaneous canine cancers and may contribute to increased cytogenetic alterations and tumour formation. Using reverse transcription and DNA amplification, we isolated p53 cDNA from normal and tumour tissue of ten canine mammary cancer patients. DNA sequencing identified p53 mutations in three of the ten patients. These included tumour-associated p53 gene mutations within exons 2 and 5 and a germ line deletion of exons 3 to 7. These results support a role for p53 inactivation in canine mammary tumour formation and breed predisposition to cancer. Such information could prove invaluable in the successful outbreeding of inherited predisposition to cancer in the dog. A putative polymorphism was also identified at codon 69 in exon 4 and we discuss the possibility that similar polymorphisms may be associated with human breast cancer.

Isolation of canine p53 cDNA and detailed characterization of the full length canine p53 protein.

The p53 tumour suppressor protein plays a central role in the maintenance of genomic integrity. Mutations of the p53 gene are found in a number of canine cancers and many contribute to tumour formation. Here we describe isolation and expression of the complete wild type canine p53 cDNA. The encoded full length canine p53 protein displays strong sequence homology with p53 proteins from other higher vertebrates. Canine p53 protein produced in vitro was shown to recognize and bind to p53-specific DNA targets derived from the p21 and GADD45 promoters and to a consensus p53 binding site. We also show that canine p53 associates with oligonucleotides representing damaged DNA sites and undergoes proteolytic cleavage similar to that described for murine and human p53 proteins. Finally, we show that the canine p53 protein is able to transcriptionally activate a p53-dependent reporter gene in vivo. The results suggest that canine p53 is similar both in structure and function to human p53 and that canine cancer may provide a useful clinical model in the search for effective anti-cancer therapies based on p53.

Mutations of the p53 gene in canine lymphoma and evidence for germ line p53 mutations in the dog.

Mutations of the p53 gene are associated with a number of non-lymphoid cancers of the dog. The present study investigates the p53 gene status within canine patients treated for primary and secondary lymphoma. Three out of eight patients exhibited p53 gene mutations. These included one patient with a germ line mutation and two patients with de novo p53 mutations associated with the secondary lymphoma. Allelic loss of the p53 gene was also observed within primary and secondary tumours of the three canine patients. The results indicate that germ line p53 mutations exist in dogs and may be involved in the known predisposition of some breeds to cancer. The presence of therapy-related p53 point mutations was found to be associated with chemoresistant secondary lymphomas. A causative role for DNA-damaging chemotherapy in de novo mutation of the p53 gene is discussed. Characterization of p53 inactivation in canine tumorigenesis may provide a valuable clinical model for assessing the efficacy and optimal therapeutic regimens of anti-cancer agents.

Effects of zinc finger mutations on the nucleic acid binding activities of Xenopus transcription factor IIIA.

Transcription factor IIIA (TFIIIA) is required for the activation of 5S RNA gene transcription as well as the storage of 5s RNA as a 7S ribonucleoprotein particle. Interaction with both nucleic acids is mediated through nine C2H2 zinc fingers. In order to determine amino acid regions necessary for nucleic acid interaction, a series of substitution mutants Xenopus laevis TFIIIA have been constructed and expressed as recombinant proteins in Escherichia coli. The mutant proteins were purified to homogeneity and analyzed for 5S RNA gene and 5S RNA binding activities using a nitrocellulose filter binding assay. All of the mutant TFIIIA proteins retained full 5S RNA binding activity. Substitution of fingers 2, 3, and 4-6 of TFIIIA with zinc finger sequences from other proteins significantly reduced the interaction of the protein with the 5S RNA gene. In contrast, substitution of finger 1 or finger 7 had little effect on the interaction of TFIIIA with the 5S RNA gene. The results of scanning substitution mutagenesis within the first three zinc fingers of TFIIIA suggested that DNA contacts made by the alpha-helical regions of finger 2 and particularly of finger 3 provide the majority of the free energy of the TFIIIA-DNA interaction. Basic amino acids found at the same position within the alpha-helices of fingers 2 and 3 of TFIIIA are required for high-affinity DNA binding activity. The identification of amino acid residues critical for the formation of a TFIIIA-DNA complex contributes to our understanding of zinc finger protein-nucleic acid interactions.

Interaction of the RNA binding fingers of Xenopus transcription factor IIIA with specific regions of 5 S ribosomal RNA.

Zinc fingers 4 to 7 of Xenopus transcription factor IIIA (TFIIIA) represent the minimal polypeptide necessary for high-affinity binding to 5 S RNA. Mutations covering the entire 5 S RNA structure have been compared for their effects on the binding affinity of full-length TFIIIA and a polypeptide consisting of fingers 4 to 7 of TFIIIA (zf4-7). In addition, ribonuclease footprinting was used to compare the binding sites of TFIIIA and zf4-7 on 5 S RNA. The consistency between the data obtained from these two approaches provided a clear indication that zinc fingers 4 to 7 of TFIIIA bind to a central core region on the 5 S RNA molecule consisting of loop B/helix II/loop A/helix V/region E. This information was used to design a truncated 75-nucleotide-long RNA molecule that retains high affinity for zf4-7. Therefore, we conclude that the specific interaction of TFIIIA with 5 S RNA can be represented by a complex formed between a four zinc finger polypeptide and a truncated 5 S RNA molecule.

Contribution of individual base pairs to the interaction of TFIIIA with the Xenopus 5S RNA gene.

The effects of a series of point mutations within the Xenopus borealis somatic-type 5S RNA gene on transcription factor IIIA (TFIIIA) binding affinity were quantified. These data define a critical sequence-dependent contact region within the classical box C promoter element from base pair 80 to 91. Substitution of GC base pairs at positions 81, 85, 86, 89, and 91 significantly reduce TFIIIA binding affinity. Base pairs located at other positions within the box C contact region provide a moderate contribution to TFIIIA-5S gene interaction. In contrast to the extensive set of sequence contacts within the box C element, TFIIIA interaction is localized primarily to two GC base pairs at positions 70 and 71 within the intermediate promoter element. A selected amplification and binding assay (SAAB) was performed with a synthetic internal control region (ICR) randomized from base pair 78 to 95 to identify box C promoter sequences bound with high affinity by TFIIIA. The wild-type 5S RNA gene sequence from 79 to 92 is strongly selected. These results are consistent with the critical role of the box C element in sequence-dependent promoter recognition by TFIIIA.

Mutations in 5S DNA and 5S RNA have different effects on the binding of Xenopus transcription factor IIIA.

The effects on TFIIIA binding affinity of a series of substitution mutations in the Xenopus laevis oocyte 5S RNA gene were quantified. These data indicate that TFIIIA binds specifically to 5S DNA by forming sequence-specific contacts with three discrete sites located within the classical A and C boxes and the intermediate element of the internal control region. Substitution of the nucleotide sequence at any of the three sites significantly reduces TFIIIA binding affinity, with a 100-fold reduction observed for substitutions in the box C subregion. These results are consistent with a direct interaction of TFIIIA with specific base pairs within the major groove of the DNA. A comparison of the TFIIIA binding data for the same mutations expressed in 5S RNA indicates that the protein does not make any strong sequence-specific contacts with the RNA. Although the protein footprinting sites on the 5S DNA and 5S RNA are coincident, nucleotide substitutions in 5S RNA which moderately reduce TFIIIA binding affinity do not correspond at all to the three specific TFIIIA interaction sites within the gene. The implications of these results for models which attempt to reconcile the DNA and RNA binding activities of TFIIIA by proposing a common structural motif for the two nucleic acids are discussed.