Isolering og karakterisering av et operon for chaperoner fra bakterien Chloroflexus aurantiacus

Genet som koder for chaperoninet GroESL1 i C. aurantiacus ble isolert fra et genomisk bibliotek av bakterien. Genet som koder for GroESL2 var tidligere isolert fra det samme biblioteket (Vanberg, Hovedfagsoppgave UiO, 2001) ved hjelp av PCR. Denne metoden ble derfor valgt. Da oppgaven ble påbegynt var ikke genomet til C. aurantiacus ferdigsekvensert, men en stor del av genomet lå ute på NCBI som ”shot-gun” sekvenser. Imidlertid var noe av sekvensen til groESL1 allerede kjent, Chlo_724 (figur 4.20) var på 1430 bp. Da promoterområdet til operonet og groES1 utgjorde 582 av disse basene, og forventet størrelse til groEL1 var ca. 2000 bp, manglet sekvensen ca. 1000 bp. Spesifikke primere for genet ble utformet og en PCR basert søkemetode etter King (1997) sammen med en noe modifisert metode etter Yu og Bloem (1996) ble brukt. Med disse metodene ble flere positive fagkloner isolert fra biblioteket. Innskuddene fra de positive fagklonene ble videre analysert med PCR med kombinasjonen av vektorspesifikke primere og genspesifikke primere for å finne ut om hele groESL1 var tilstede i innskuddet. Sekvenseringen av innskuddene ble gjort av firmaene GATC og MWG. Flere innskudd ble isolert og sekvensert, men den fullstendige sekvensen til groESL1 ble ikke funnet. En klon, kalt Plakk2, ga en fortsettelse av sekvensen Chlo_724 på ca. 800 bp. Denne sekvensen ble analysert i programmet WebCutter. I dette programmet ble sekvensen kuttet in silico med restriksjonsenzymet Sau3AI, som var enzymet som var brukt til å kutte genomisk DNA fra C. aurantiacus da biblioteket ble konstruert. Resultatet av analysen viste 17 kutteseter i området der sekvensene sluttet. Høsten 2004 ble genomet til C. aurantiacus ferdig sekvensert og lagt ut på databasene til NCBI som en ”shot gun” sekvens i form av ”contigs”, delsekvenser. Siste del av Plakk2, omtrent 200 bp, ble brukt i BLAST søk. En klon, Chlo01_574 viste 100 % sekvenslikhet med den siste delen av Plakk2. Sekvensen som ligger mellom det som var kjent fra før og den siste delen av Plakk2 var imidlertid ikke å finne i databasen. Muligens var det her et glipp i sekvenseringen fra TIGR. Sekvensen ble satt sammen så de utgjorde hele operonet groESL1 (figur 4.24). Sekvensen til genet var på 2228 bp og inneholdt stopp-kodonet TAA. Sekvensen ble oversatt til proteinsekvens og molekylvekten beregnet ved hjelp av programmet ExPacy. Molekylvekten på dette proteinet var 59,7 kDa. Det ble utformet primere for å isolere hver av de to genene i operonet, groES1 og groEL1, ved hjelp av PCR. Primersettene ble brukt både med kromosomalt DNA fra og biblioteket som templat. Primersettet til groES1 ga gode resultater med begge templatene, primersettet til groEL1 ga kun resultat med kromosomalt DNA som templat. Dette styrket teorien om at hele sekvensen til groEL1 ikke var å finne i en klon. PCR produktene ble sekvensert og viste seg å stemme med sekvensene i operonet. De to genene skulle uttrykkes og ble derfor satt inn i hver sin pET vektor som ble brukt til å transformere BL21 Star celler. Det viste seg at det bare var groES1 som ble uttrykt på denne måten. Sekvensen til groEL1 ble videre analysert i programmet Rare Codon Calculator der det ble funnet at dette genet hadde i alt 14 sjeldne kodoner for aminosyrene prolin og arginin. Sjeldne kodoner kan være årsaken til at genet ikke blir uttrykt, eller at det blir for tidlig terminering av heterologe gener. Det ble derfor forsøkt å uttrykke groEL1 i vertscellen BL21-CodonPlus (DE3) RP. Stammen BL21-CodonPlus (DE3) RP inneholder ekstra kopier av argU og proL, genene som koder for tRNA som gjenkjenner kodonene AGA og AGG for arginin og kodonet CCC for prolin. Da IPTG indusert celleekstrakt fra disse cellene ble analysert på SDS-PAGE, viste det seg at et protein på 60 kDa var uttrykt

Morphological and molecular characterization of developing vertebral fusions using a teleost model

<p>Abstract</p> <p>Background</p> <p>Spinal disorders are a major cause of disability for humans and an important health problem for intensively farmed animals. Experiments have shown that vertebral deformities present a complex but comparable etiology across species. However, the underlying molecular mechanisms involved in bone deformities are still far from understood. To further explicate the mechanisms involved, we have examined the fundamental aspects of bone metabolism and pathogenesis of vertebral fusions in Atlantic salmon (<it>Salmo salar</it>).</p> <p>Results</p> <p>Experimentally, juvenile salmon were subjected to hyperthermic conditions where more than 28% developed fused vertebral bodies. To characterize the fusion process we analyzed an intermediate and a terminal stage of the pathology by using x-ray, histology, immunohistochemistry, real-time quantitative PCR and <it>in situ </it>hybridization. At early stage in the fusion process, disorganized and proliferating osteoblasts were prominent at the growth zones of the vertebral body endplates. PCNA positive cells further extended along the rims of fusing vertebral bodies. During the developing pathology, the marked border between the osteoblast growth zones and the chondrocytic areas connected to the arches became less distinct, as proliferating cells and chondrocytes blended through an intermediate zone. This cell proliferation appeared to be closely linked to fusion of opposing arch centra. During the fusion process a metaplastic shift appeared in the arch centra where cells in the intermediate zone between osteoblasts and chondrocytes co-expressed mixed signals of chondrogenic and osteogenic markers. A similar shift also occurred in the notochord where proliferating chordoblasts changed transcription profile from chondrogenic to also include osteogenic marker genes. In progressed fusions, arch centra and intervertebral space mineralized.</p> <p>Conclusion</p> <p>Loss of cell integrity through cell proliferation and metaplastic shifts seem to be key events in the fusion process. The fusion process involves molecular regulation and cellular changes similar to those found in mammalian deformities, indicating that salmon is suitable for studying general bone development and to be a comparative model for spinal deformities.</p

Korišćenje in vitro model sistema za proučavanje osnovnih bioloških procesa kod riba

With the increasing replacement of fish meal and fish oil with new ingredients in aquaculture diets, imbalances in amino acids, fatty acids, vitamins and minerals can occur. The metabolic and regulatory processes underlying these nutrition-induced imbalances in fish are still not fully understood. At the cellular level, essential dietary compounds and micro-nutrients have been shown to influence lineage determination, differentiation and proliferation of certain cell types, and hence the development of tissue structures and organogenesis. An improved understanding of cellular and molecular events occurring during development in teleosts will enable us to better characterize and define particular requirements, customize feed components, and thus enable development of sustainable feeds, minimize the occurrence of disorders as well as maintain the continuous growth of the fish. In vitro techniques have a great potential in experiments involving mechanism based hypothesis testing, where there is a significant need for a complete understanding of basic biological processes. Cell cultures provide means to study single-factor effects and the combinations thereof in detail, and further, to investigate the role of particular nutrients and their specific gene interactions, which are not possible when working at the organism level. In addition, whole organisms are complex and vary individually, depending on age, sex, health status, type of meal, genetics etc., which makes it difficult to accurately simulate nutritional processes. In vitro experiments offer the unique opportunity to develop standardized methods to study quality of novel and fortified feed products by studying the cellular and molecular effect of different types of food products, ranging from proteins to n-3 fatty acids (FAs) and from fat-soluble vitamins to minerals and trace elements. The system may also be used for studies on the development of functional feeds such as probiotics, prebiotics, bioactive peptides, lipase inhibitors, fat and cholesterol binders and antioxidants. Results from selected in vitro trials, showing how different nutrients may influence development of fat, bone and muscle cells and lipid metabolism in liver cells, will be presented.Zamena ribljeg brašna i ulja sa novim, alternativnim izvorima proteina i masti u hrani za ribe može dovesti do narušavanja odnosa amino kiselina, masnih kiselina i minerala kod ovih organizama. Promene u metaboličkim i regulatornim procesima koje mogu da nastanu loše balansiranom ishranom još uvek nisu dobro proučene kod riba. Esencijalne hranljive materije i mikronutrijenti mogu da utiču na razvoj pojedinih ćelijskih linija, njihovu proliferaciju, a samim tim i na pravilan razvoj tkiva i organa. Bolje razumevanje ćelijskih i molekularnih procesa, koji se dešavaju tokom razvića košljoriba će nam omogućiti da bolje razumemo i definišemo određene zahteve za ishranom na svakom stupnju razvoja riba, prilagodimo komponente riblje hrane, a samim tim i omogućimo normalan razvoj i rast riba, kao i da sprečimo pojavu različitih poremećaja. Korišćenje in vitro sistema predstavlja veliki potencijal za testiranje novih komponenata hrane i daje nam mogućnost za razumevanje osnovnih bioloških procesa kod riba. Prednost korišćenja ćelijskih kultura je što nam one omogućavaju da proučavamo uticaj pojedinačnih faktora, ali i kombinaciju dva ili više različitih faktora i njihov uticaj na gene, što nije moguće istraživati na nivou organizma. Pored toga proučavanje organizma kao celine je jako složeno, jer zavisi od pola, zdravstvenog stanja jedinke, ishrane, genetike, itd., te je na ovaj način lakše simulirati procese ishrane. In vitro eksperimenti nude jedinstvenu priliku da razvijemo standardizovane metode za proučavanje kvaliteta i efekata novih hranljivih materija: od proteina, preko masnih kiselina i minerala, do elemenata, koji se u hrani nalaze u tragovima. Ovi sistemi mogu se koristiti i za proučavanje novih, funkcionalnih hraniva, kao što su: probiotici, prebiotici, bioaktivni peptidi, masti, inhibitori lipaze, antioksidansi. Biće prikazani rezultati odabranih in vitro ispitivanja uticaja različitih hraniva na metabolizam masnog tkiva, kostiju i mišića kao i na sam metabolizam masti

Molecular pathology of vertebral deformities in hyperthermic Atlantic salmon (Salmo salar)

<p>Abstract</p> <p>Background</p> <p>Hyperthermia has been shown in a number of organisms to induce developmental defects as a result of changes in cell proliferation, differentiation and gene expression. In spite of this, salmon aquaculture commonly uses high water temperature to speed up developmental rate in intensive production systems, resulting in an increased frequency of skeletal deformities. In order to study the molecular pathology of vertebral deformities, Atlantic salmon was subjected to hyperthermic conditions from fertilization until after the juvenile stage.</p> <p>Results</p> <p>Fish exposed to the high temperature regime showed a markedly higher growth rate and a significant higher percentage of deformities in the spinal column than fish reared at low temperatures. By analyzing phenotypically normal spinal columns from the two temperature regimes, we found that the increased risk of developing vertebral deformities was linked to an altered gene transcription. In particular, down-regulation of extracellular matrix (ECM) genes such as <it>col1a1</it>, <it>osteocalcin</it>, <it>osteonectin </it>and <it>decorin</it>, indicated that maturation and mineralization of osteoblasts were restrained. Moreover, histological staining and <it>in situ </it>hybridization visualized areas with distorted chondrocytes and an increased population of hypertrophic cells. These findings were further confirmed by an up-regulation of <it>mef2c </it>and <it>col10a</it>, genes involved in chondrocyte hypertrophy.</p> <p>Conclusion</p> <p>The presented data strongly indicates that temperature induced fast growth is severely affecting gene transcription in osteoblasts and chondrocytes; hence change in the vertebral tissue structure and composition. A disrupted bone and cartilage production was detected, which most likely is involved in the higher rate of deformities developed in the high intensive group. Our results are of basic interest for bone metabolism and contribute to the understanding of the mechanisms involved in development of temperature induced vertebral pathology. The findings may further conduce to future molecular tools for assessing fish welfare in practical farming.</p

A full factorial design to investigate interactions of variable essential amino acids, trace minerals and vitamins on Atlantic salmon smoltification and post transfer performance

To contribute in knowledge for the development of safe, efficient and sustainable functional salmon diets, we ran a feeding trial applying a 23 full factorial design to investigate combined effects, on Atlantic salmon smoltification and post transfer performance, of variable supplementation levels of essential amino acid (Lys, Met, Thr and Arg), essential trace mineral (Zn, Fe and Se) and vitamins (E, C and astaxanthin as provitamin A) premixes in low fishmeal diets, using crystalline amino acids, organic trace minerals and synthetic vitamins, respectively. The nutrient levels used in our study were chosen to meet the known requirements of fish reflecting the variation in commercial feeds. Fish performance, nutrient digestibility, skin, and intestinal health were evaluated in Atlantic salmon parr-smolt, the latter by means of qPCR, global transcriptomics, and immunohistochemistry. The results revealed the potential for significant improvement of salmon post smoltification growth by simultaneous dietary level increase of Met, Lys, Thr and Arg (5% higher body weight increase). Significantly negative effect on fish post transfer growth and survival (22.5 % lower body weight growth and 2.6 times higher mortality) was observed in the high dietary vitamin supplementation treatments which was not present in the simultaneous high trace mineral and vitamin supplementation treatments (8% higher body weight increase and 2.8 times lower mortality in the high trace mineral supplementation treatments). In the high trace mineral supplemented dietary treatments was also observed improved FCR (8.5 %) and a further improvement in performance was seen in the treatments with simultaneous high essential amino acid and trace mineral supplementation levels (12.6 % higher body growth increase). Redox-sensitive gene and extracellular matrix components’ gene transcription effects and compensatory mechanisms on protein and energy metabolism, immune modulation, skin repair systems and erythropoiesis were observed by transcriptomic and histological analyses in response to the variable dietary essential nutrient levels.publishedVersio

The skin mucosal barrier of lumpfish (Cyclopterus lumpus L.) is weakened by exposure to potential aquaculture production related stressors

Various cleaner fish species, such as the lumpfish (Cyclopterus lumpus L.), are used in the sea cage production of Atlantic salmon (Salmo salar L.) as a control measure against the ectoparasitic salmon louse (Lepeophtheirus salmonis). However, during severe lice infestation, alternative treatments are required to control parasitic burden. The aim of this study was to gain insight into how lumpfish skin responds to different chemicals used to treat parasites. We collected skin from lumpfish from both research facilities (tank reared fish) and commercial production (cage reared fish), and used operational welfare indicators (OWIs), in vitro models, histology and transcriptomics to study how the skin responded to two anti-parasitic oxidative chemicals, hydrogen peroxide (H2O2) and peracetic acid (PAA). Lumpfish sampled from the farm were classified as clinically healthy or weak according to their morbidity status, and fish from each category were used to gain insight into how the therapeutics affect the skin barrier. Differences between healthy and weakened (moribund) fish, and between treated fish from each of the two groups, were observed. Histological examination showed an overall reduced skin quality in fish characterized as moribund, including different grades of exposed bony plates. In vitro oxidant-treated lumpfish skin had reduced migration capacity of keratocytes, a weakened epidermal barrier and altered gene transcription, changes that are known predisposing factors to secondary infections. Skin from non-treated, healthy fish sampled from commercial farms exhibited similar features and attributes to oxidant-exposed tank reared fish from a research facility, suggesting that apparently healthy cage-held lumpfish exhibited stress responses in the epidermal barrier. The results of the study outline the risks and consequences lumpfish can face if accidentally subjected to potential anti-parasitic oxidant treatments aimed at Atlantic salmon. It also strengthens the evidence behind the requirement that lumpfish should be removed from the cages before being potentially exposed to this type of treatment and outlines the potential risks of differing husbandry practices upon lumpfish health, welfare and resilience.The skin mucosal barrier of lumpfish (Cyclopterus lumpus L.) is weakened by exposure to potential aquaculture production related stressorspublishedVersio

Welfare and performance of ballan wrasse (Labrus bergylta) reared at two different temperatures after a preparatory feeding trial with enhanced dietary eicosapentaenoic acid

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Nasal responses to elevated temperature and Francisella noatunensis infection in Atlantic cod (Gadus morhua)

We report the histological and transcriptomic changes in the olfactory organ of Atlantic cod exposed to Francisella noatunensis. Experimental infection was performed at either 12oC or 17oC. Infected fish presented the classic gross pathologies of francisellosis. Nasal morpho-phenotypic parameters were not significantly affected by temperature and infection, except for the number of mucus cells in the 12oC group seven weeks after the challenge. A higher number of genes were altered through time in the group reared at 17oC. At termination, the nasal transcriptome of infected fish in both groups was similar to the control. When both infected groups were compared, 754 DEGs were identified, many of which were involved in signalling, defence, transmembrane and enzymatic processes. In conclusion, the study reveals that elevated temperature could trigger responses in the olfactory organ of Atlantic cod and shape the nasal response to F. noatunensis infection.Nasal responses to elevated temperature and Francisella noatunensis infection in Atlantic cod (Gadus morhua)publishedVersio

Evaluating a crowding intensity scale and welfare indicators for Atlantic salmon in sea cages

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