Genetic profile of scrapie codons 146, 211 and 222 in the PRNP gene locus in three breeds of dairy goats

Polymorphisms at PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles (146S, 146D, 211Q and 222K) were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds

Genetic profile of scrapie codons 146, 211 and 222 in the PRNP gene locus in three breeds of dairy goats

Polymorphisms at PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles (146S, 146D, 211Q and 222K) were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds

Molecular characterization of a gilthead sea bream (Sparus aurata) muscle tissue cDNA for carnitine palmitoyltransferase 1B (CPT1B)

Understanding the control of piscine fatty acid metabolism is important for determining the nutritional requirements of fish, and hence for the production of optimal aquaculture diets. The regulation and expression of carnitine palmitoyltransferase 1 (CPT1; EC No 2.3.1.21) are critical processes in the control fatty acid metabolism, and here we report a cDNA from gilthead sea bream (Sparus aurata) which encodes a protein with high identity to vertebrate CPT1. This sea bream CPT1 mRNA is predominantly expressed in skeletal and cardiac muscle, with little expression in other tissues. Phylogenetic analysis of other vertebrate CPT1 sequences show that fish genomes contain a single gene related to mammalian CPT1B, and a further two multi-gene families related to mammalian CPT1A. Genes related to mammalian CPT1C are absent in fish. Therefore, based on both functional and evolutionary orthology to mammalian CPT1B, the sea bream CPT1 reported here is a CPT1B isoform. Sea bream CPT1B mRNA expression progressively decreases in heart and muscle up to 12 hours after last feeding, but returns to initial, non-fasted levels after 72 hours. In contrast, in liver non-fasted expression is low, but strongly increases at 24 and 72 hours after last feeding. In white muscle and liver, CPT1B mRNA expression is highly correlated with the expression of peroxisomal proliferator-activated receptor ı (PPARı).Thus fatty acid metabolism by CPT1B and its control by PPARs is similar in fish and mammals, but multiple genes for CPT1A-like proteins in fish also suggest different and more complex pathways of lipid utilisation than in mammals

Piscine UDP-glucuronosyltransferase 1B

Glucuronidation is an important detoxification pathway for organic pollutants in fish. We report here the isolation and characterisation of UDP-glucuronosyltransferases (UGT) genes from the closely related marine flatfish, plaice (Pleuronectes platessa) and flounder (Platichthys flesus). The deduced amino acid sequences share greater similarity with mammalian UGT1 family genes than UGT2 genes (44-47% and 39-40% amino acid identity respectively) and have been designated UGT1B. Both plaice and flounder UGT1B mRNAs are most highly expressed in liver, but are also expressed in intestine, gill, kidney and adipose tissue to a greater extent than muscle, heart or brain. Plaice UGT1B mRNA was undetectable in gametes or fertilised eggs and there was a large increase in expression between gastrulation and myotome formation after which levels declined some 5-10 fold. Flounder UGT1B mRNA was increased in liver after intraperitoneal injection of Arochlor 1254 or lindane, but not after perflourooctanoic acid or 3-methylcholanthrene. In isolated flounder hepatocytes UGT1B mRNA was increased by benzo(a)pyrene but not by ethynylestradiol. Expression of a cDNA for plaice UGT1B in cos7 cells resulted in higher 1-naphthol conjugation in cell homogenates compared to steroid conjugation, whilst bilirubin and bile acid conjugation undetectable. This indicates that the plaice gene codes for the phenol-conjugating UGT previously purified in our laboratory from this species and that it is likely to play a major role in the detoxification of polyaromatic hydrocarbons in flatfish. Its role in development is unknown. UGT1B genes are also present in pufferfish (Tetraodon nigroviridis) and zebrafish (Danio rerio) genomes, but that they differ in their genic organisation. Pufferfish possess multiple (repeated) complete UGT1 genes and Southern blots indicate that the homologous plaice UGT1B gene may also be organised in this way. In contrast, zebrafish appear to have two UGT1 loci whose sequences and intron/exon structures are closely related to that of plaice, however, the organisation of these genes is similar to the mammalian UGT1 family since each has multiple repeated exon 1’s which are alternatively spliced to a common set of exons encoding the aglycone binding domain. Taken together with evidence from phylogenetic comparison of fish sequences with UGT1 and UGT2 families in mammals, we suggest these homologous fish UGTs should all be included within the vertebrate UGT1 family and designated as UGT1B

Hepatic gene expression in flounder chronically exposed to multiply polluted estuarine sediment: Absence of classical exposure ‘biomarker’ signals and induction of inflammatory, innate immune and apoptotic pathways

The effects of chronic long term exposure to multiply-polluted environments on fish are not well understood, but environmental surveys suggest that such exposure may cause a variety of pathologies, including cancers. Transcriptomic profiling has recently been used to assess gene expression in European flounder (Platichthys flesus) living in several polluted and clean estuaries. However, the gene expression changes detected were not unequivocally elicited by pollution, most likely due to the confounding effects of natural estuarine ecosystem variables. In this study flounder from an uncontaminated estuary were held on clean or polluted sediments in mesocosms, allowing control of variables such as salinity, temperature, and diet. After 7 months flounder were removed from each mesocosm and hepatocytes prepared from fish exposed to clean or polluted sediments. The hepatocytes were treated with benzo(a)pyrene (BAP), estradiol (E2), copper, a mixture of these three, or with the vehicle DMSO. A flounder cDNA microarray was then used to measure hepatocyte transcript abundance after each treatment. The results show that long term chronic exposure to a multiply-polluted sediment causes increases in the expression of mRNAs coding for proteins of the endogenous apoptotic program, of innate immunity and inflammation. Contrary to expectation, the expression of mRNAs which are commonly used as biomarkers of environmental exposure to particular contaminants were not changed, or were changed contrary to expectation. However, acute treatment of hepatocytes from flounder from both clean and polluted sediments with BAP or E2 caused the expected changes in the expression of these biomarkers. Thus transcriptomic analysis of flounder exposed long-term to chronic pollution causes a different pattern of gene expression than in fish acutely treated with single chemicals, and reveals novel potential biomarkers of environmental contaminant exposure. These novel biomarkers include Diablo, a gene involved in apoptotic pathways and highly differentially regulated by both chronic and acute exposure to multiple pollutants

Study of peroxisome proliferator-activator receptors - PPARs in farmed fish

My thesis was part of a European research project which intention was the study of transcription control of lipid metabolism in farmed fish and especially in sea bream (Sparus aurata), sea bass (Dicentrarchus labrax), salmon (Salmo salar) and plaice (Pleuronectes platessa). For this purpose we initially performed the cloning and analysis of PPARs in these species. PPARs are ligand-inducible transcription factors belonging to the nuclear hormone superfamily. We also studied PPARs’ tissue expression profile and the properties of their two basic domains DBD and LBD. At the beginning, we isolated and cloned parts of the three PPAR isotypes genes, α, β and γ from both species and part of the second isoform of sea bream PPARα gene. Sequence analysis of these genomic fragments revealed significant homology with the mammalian PPAR genes. To confirm that these isolated genomic fragments constitute parts of functional genes we also isolated and cloned the cDNAs of sea bream and sea bass PPARs. We amplified with RT-PCR their coding regions as well as parts of the 3’ and 5’ untranslated regions. The amino acid sequences of fish PPARs also revealed high homology with mammalian and amphibian homologs. The expression profile of PPARs was determined by the RNase protection assay. The results demonstrate that sea bream and sea bass PPARs present similarities and differences with the mammalian PPARs pattern. This maybe indicates the physiological variations between fish and mammals. Using the Electrophoretic Mobility Shift Assay (EMSA) we studied the abilities of sea bream, sea bass, plaice and salmon PPARs to form heterodimers with RXR and recognize and bind to well-characterized or potential PPAR response elements. The fish PPARs were able to form heterodimers with RXR and bind to DR-1 response elements we tested. Thus, the properties of PPARs’ DBD and LBD domains are conserved in lower evolutionally vertebrates. Finally, through CoActivator-Dependent Receptor Ligand Assay (CARLA) we identified compounds that can act as ligands of fish PPARs. The results suggest that fish, except salmon, PPAR α and β demonstrate similar properties with the mammalian counterparts, sharing common ligands. In contrast, the fish PPARγ exhibit low or no affinity for any of the compounds tested. According to the results presented here, the fish PPARs are structurally homologs with their mammalian counterparts and possibly they are involved in similar functions controlling the expression of relevant genes.Η παρούσα διατριβή πραγματοποιήθηκε στα πλαίσια ευρωπαϊκού προγράμματος με στόχο τη μελέτη του μεταγραφικού ελέγχου του μεταβολισμού των λιπιδίων σε καλλιεργούμενα είδη ψαριών, όπως στην τσιπούρα (Sparus aurata), στο λαβράκι (Dicentrarchus labrax), στο σολομό (Salmo salar) και στο φασί του Ατλαντικού (Pleuronectes platessa). Για το σκοπό πραγματοποιήθηκε αρχικά κλωνοποίηση και ανάλυση των PPARs που αποτελούν μεταγραφικούς παράγοντες της υπεροικογένειας των πυρηνικών ορμονικών υποδοχέων και ενεργοποιούνται από λιπαρά οξέα και φαρμακευτικές ουσίες με υπολιπιδαιμική δράση. Στη συνέχεια πραγματοποιήθηκε μελέτη του προτύπου έκφρασης και μελέτη των ιδιοτήτων των δυο βασικότερων περιοχών, DBD (DNA binding domain) και LBD (Ligand binding domain) των υποδοχέων. Αρχικά, επιτεύχθηκε η κλωνοποίηση τμημάτων των γονιδίων των τριών τύπων PPAR α, β και γ, από την τσιπούρα και το λαβράκι, καθώς και τμήμα του γονιδίου της δεύτερης ισομορφής του PPARα από την τσιπούρα. Διαπιστώθηκε σημαντική ομοιότητα της αλληλουχίας και της οργάνωσης των γονιδίων των PPARs των ψαριών με τα ομόλογα γονίδια των θηλαστικών και των αμφιβίων. Στη συνέχεια, προχωρήσαμε σε κλωνοποίηση και των cDNAs των PPARs από τα συγκεκριμένα είδη, ώστε να διαπιστωθεί εάν οι συγκεκριμένες γενωμικές ακολουθίες αποτελούν λειτουργικά γονίδια των ψαριών. Με RT-PCR ενισχύθηκαν οι κωδικοποιούμενες περιοχές και τμήματα των 3’ και 5’ αμετάφραστων άκρων των PPARs από την τσιπούρα και το λαβράκι. Και στην περίπτωση αυτή διαπιστώθηκαν πολύ υψηλά ποσοστά ομοιότητας μεταξύ των αμινοξικών αλληλουχιών των PPARs των ψαριών με τους ομόλογους τύπους των θηλαστικών και των αμφιβίων. Η μελέτη του προτύπου της ιστοειδικής έκφρασης των PPARs στην τσιπούρα και στο λαβράκι, με τη μέθοδο RNase protection, έδειξε ότι η έκφρασή τους παρουσιάζει αρκετές ομοιότητες αλλά και σημαντικές διαφορές με αυτή των θηλαστικών, υποδηλώνοντας διαφοροποιήσεις στη φυσιολογία τους. Με τη μέθοδο της ηλεκτροφορητικής κινητικότητας των συμπλόκων DNA- πρωτεϊνών (EMSA-Electrophoretic Mobility Shift Assay) μελετήθηκε η ικανότητα των PPARs από την τσιπούρα, το λαβράκι, το φασί του Ατλαντικού και το σολομό, να προσδένονται ως ετεροδιμερή με τους υποδοχείς RXR σε ακολουθίες που αποτελούν γνωστά ή υποτιθέμενα αποκρινόμενα στοιχεία των PPARs. Οι υποδοχείς και από τα τέσσερα είδη ψαριών σχημάτισαν ετεροδιμερή με τους υποδοχείς RXR και αναγνώρισαν τις ακολουθίες των αποκρινόμενων στοιχείων του τύπου DR-1 που εξετάστηκαν, αποδεικνύοντας τη συντήρηση των ιδιοτήτων τόσο της DBD περιοχής όσο και της LBD περιοχής και σε κατώτερα εξελικτικά σπονδυλωτά. Με τη μέθοδο CARLA (CoActivator-Dependent Receptor Ligand Assay) τέλος εντοπίστηκαν ουσίες που μπορούν να αποτελέσουν προσδέτες των PPARs των ψαριών. Διαπιστώθηκε ότι οι LBD περιοχές των PPAR α και β των ψαριών εκτός του σολομού, παρουσιάζουν παρόμοιες ιδιότητες με αυτές των θηλαστικών, αναγνωρίζοντας αρκετές από τις ουσίες που εξετάστηκαν, ενώ η αντίστοιχη περιοχή του PPARγ παρουσιάζει χαμηλή ή και καθόλου συγγένεια για τις συγκεκριμένες ουσίες. Σύμφωνα με τα αποτελέσματα της παρούσας εργασίας, οι PPARs των ψαριών είναι δομικά ομόλογοι με τους PPARs των θηλαστικών και πιθανότατα συμμετέχουν σε ανάλογες λειτουργίες ρυθμίζοντας την έκφραση των αντίστοιχων γονιδίων

Indigenous Lactic Acid Bacteria Isolated from Raw Graviera Cheese and Evaluation of Their Most Important Technological Properties

The aim of the present study was to characterize LAB isolates from raw-milk cheeses, to evaluate some of their technological properties and to select a few ‘wild’ LAB strains that could potentially be used as starter cultures. LAB strains were isolated and identified from raw milk, curd, and cheese at 30, 60, and 90 days of ripening. A total of 100 strains were isolated, 20 from each phase of ripening. All isolates were tested for acidification ability, curd formation, and aroma production at 32 °C and 42 °C after 24 and 48 h. Following the acidification test, 42 strains were selected for identification and characterization of their technological properties. A high proportion of lactic acid bacteria and Gram + cocci were found throughout the cheese-making process. Enterococci reached their maximum proportion on the 7th day of ripening while Lactobacilli increased significantly during the first month of ripening. Forty-two strains were identified by phenotypic, biochemical, and molecular techniques. Lactococci were predominant in raw milk and curd while Lactobacilli in the ripening of the cheese. Four LAB strains including one Leuconostoc pseudomenteroides, two Lacticaseibacillus paracasei subsp. paracasei and one Enterococcus hirae, were proposed for their potential use as starters or secondary cultures