Transcript detection of the AHR gene in the dog
Jong, T. de
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Summary Introduction: the Aryl Hydrocarbon Receptor is best known for its function in regulating xenobiotic metabolism and dioxin toxicity.  AHR activation by environmental contaminants is considered to be an adaptive response, which could decrease the toxicity of these environmental contaminants. On the other hand, activation of AHR could also mediate the toxicity of environmental contaminants. AHR mRNA is expressed in a variety of human tissues. Highest expression is detected in the placenta and in liver, while in pancreas, heart and lung expression is also relatively high.  Research has shown that AHR-deficiency in mice resulted in a loss of teratogenesis caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and susceptibility to chemical carcinogenesis by benzo(a)pyrene.  So AHR plays a role in tumour promotion/initiation, while recent evidence suggests AHR also plays a role in tumour progression.  On the other hand, AHR deficient mice have a reduced body weight, display reduced fecundity and are defective in development of both liver and immune system. In these mice fetal vascular structures are found in the eyes, kidneys and liver.  Objective: to study the conservation of the AHR gene and investigating whether there is more than just one transcript of the AHR gene in the dog. Hypotheses: two hypotheses were formulated, namely: - The AHR gene is highly conserved. - There are multiple transcripts of the AHR gene in the dog Conservation: the protein coding transcripts of human, dog, mouse, rat, horse, chicken, rabbit and zebrafish were compared by blasting them in NCBI Blast. Both nucleotide sequences and aminoacid sequences were compared. Query coverages ranging from 47% to 91% were found taking both methods into account. Materials and Methods: cDNA from liver tissue was used for the detection of other AHR transcripts by using different primersets to perform PCR, gel electrophoresis and sequencing. Based on these sequences primers specific for the new transcript were designed. cDNA from liver, brain tissue, bone tissue, mammary tissue, placenta, pancreas, adrenal gland, uterus, ovary, kidneys and leucocytes was used to perform PCR, gel electrophoresis and qPCR to detect the presence of this new transcript (and original transcript) in these tissues. The original transcript was also sequenced. All sequencing results were blasted to the dog genome and the annotated original transcript of the AHR gene. Last, western blotting was performed to see if both the original and new transcript are translated to proteins in liver cells. Results: a smaller transcript was detected in our liver cDNA samples. Based on the found sequences specific primers were designed to confirm the existence of the new transcript. A part of exon 2 and 7 and exon 3 till 6 are not present in the new transcript. The qPCR data showed differences between the original and new transcript in certain tissues. The sequencing results for the original transcript showed that the annotated intron 10 is coding. The western blotting showed many non-specific bands so more research needs to be done before these bands can be distinguished from bands of the AHR transcripts. Discussion: even though the existence of another transcript has definitely been proven and the presence in many tissues has been detected, the relative level of expression remains to be measured in the different tissues. The possibility of this transcript undergoing nonsense mediated decay also needs further research. For a more complete picture of the conservation of the AHR gene, more species’ nucleotide sequence and aminoacid sequence could be compared. Conclusion: the first hypothesis, that the AHR gene is a highly conserved gene, should be rejected as query coverages ranging from 47% to 91% were found taking both comparing methods into account. Even though 91% is high, 47% is very low so the AHR gene is not highly conserved. The second hypothesis, that there are multiple transcripts of the AHR gene in the dog, should be confirmed based on our sequencing results and qPCR data. The new transcript probably consists of exon 1, 2, 7, 8, 9 and 10, with a premature stop codon in exon 7. Furthermore, the original transcript of the dog consists of 11 exons, instead of the annotated 12 exons.