1101. Extraction of DNA from different sample types - a practical approach for GMO testingJana Žel, Tina Demšar, Dejan Štebih, Mojca Milavec, Kristina Gruden, 2015, izvirni znanstveni članek Povzetek: Current methods based on DNA targets for the detection, identification and quantification of genetically modified organisms (GMOs) involve extraction of the DNA. Different extraction procedures have been developed for the great variety of samples from food, feed, seeds and particular plant parts. This makes the operation of routine analytical laboratories complex and workloads heavy. Here we present a decision-making system, developed over many years of GMO testing on different samples, that result in the application of only a few extraction methods for the majority of samples. Developed decision-making system enables quicker and more cost effective testing of GMOs. In addition, the performance of DNA extraction resulting from the use of the selected extraction methods is presented for use in subsequent testing of GMOs by real time PCR methods. This approach can be used as a model for similar systems based on nucleic acid analysis in food, feed, seeds and plants.
Ključne besede: extraction methods, genetically modified organisms, GMO, decision- making system (biology), GMO testing, cetyltrimethylammonium bromide, CTAB
Objavljeno v DiRROS: 29.07.2024; Ogledov: 323; Prenosov: 202
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1102. Patterns in invertebrate drift from an alpine karst aquifer over a one year periodMaja Opalički, 2015, izvirni znanstveni članek Povzetek: Patterns in invertebrate drift in the alpine karst Lipnik spring (Julian Alps, Slovenia) were investigated over a one year period. Monthly samplings of one permanent and two temporary springs, and one sampling of a spring brook benthos, yielded 23 Copepoda and Ostracoda species. More species (12) were found in the permanent than in the temporary springs (8 and 7), Elaphoidella phreatica (Sars, 1862) being the most frequent in all of them. No correlation was observed between precipitation and drift densities or the numbers of species in the permanent spring. In the temporary springs there were correlations between precipitation and the Shannon diversity index, equitability and changes in community composition. Conductivity of the water was the only physical parameter in the springs that correlated with precipitation. Species accumulation curves reached the asymptotes in all the springs but not for benthos.
Ključne besede: alpine karst aquifer, groundwater, drift
Objavljeno v DiRROS: 29.07.2024; Ogledov: 338; Prenosov: 196
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1103. Additional record of the non-indigenous copepod Pseudodiaptomus marinus (Sato, 1913) in the Adriatic SeaDavor Lučić, Patricija Mozetič, Janja Francé, Petra Lučić, Lovrenc Lipej, 2015, izvirni znanstveni članek Povzetek: The Indo-Pacific egg-carrying copepod Pseudodiaptomus marinus Sato, 1913 was recorded for the first time in the Port of Koper, Slovenia (Gulf of Trieste) in February 2015. This is the fourth finding of this species in the Mediterranean Sea, and the third for the Adriatic Sea. A rather high abundance of 73 ind. m-3 was recorded, which is considerably higher than previous findings in the Adriatic. Maritime transport is presumed to be the main cause of its introduction, primarily through ballast water release. Repeat sampling in May confirmed the presence of this copepod, indicating the possibility that the species has established a stable population in the Port of Koper.
Ključne besede: copepods, Adriatic Sea, mesozooplankton, ballast waters
Objavljeno v DiRROS: 29.07.2024; Ogledov: 328; Prenosov: 123
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1104. Improvement of root architecture under abiotic stress through control of auxin homeostasis in Arabidopsis and Brassica cropsBranka Salopek-Sondi, Stephan Pollmann, Kristina Gruden, Ralf Oelmüller, Jutta Ludwig-Müller, 2015, izvirni znanstveni članek Povzetek: Auxin plays an important role in many aspects of plant development including stress responses. Here we briefly summarize how auxin is involved in salt stress, drought (i.e. mostly osmotic stress), waterlogging and nutrient deficiency in Brassica plants. In addition, some mechanisms to control auxin levels and signaling in relation to root formation (under stress) will be reviewed. Molecular studies are mainly described for the model plant Arabidopsis thaliana, but we also like to demonstrate how this knowledge can be transferred to agriculturally important Brassica species, such as Brassica rapa, Brassica napus and Brassica campestris. Moreover, beneficial fungi could play a role in the adaptation response of Brassica roots to abiotic stresses. Therefore, the possible influence of Piriformospora indica will also be covered since the growth promoting response of plants colonized by P. indica is also linked to plant hormones, among them auxin.
Ključne besede: auxin, root development, abiotic stress
Objavljeno v DiRROS: 29.07.2024; Ogledov: 345; Prenosov: 144
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1105. Transcriptome study and identification of potential marker genes related to the stable expression of recombinant proteins in CHO clonesAndrej Blejec, Marjanca Blas, Petra Nikolić, Dominik Gaser, Kristina Gruden, Aleš Belič, Špela Baebler, Uroš Jamnikar, Andrej Francky, Holger Laux, 2015, izvirni znanstveni članek Povzetek: Background
Chinese hamster ovary (CHO) cells have become the host of choice for the production of recombinant proteins, due to their capacity for correct protein folding, assembly, and posttranslational modifications. The most widely used system for recombinant proteins is the gene amplification procedure that uses the CHO-Dhfr expression system. However, CHO cells are known to have a very unstable karyotype. This is due to chromosome rearrangements that can arise from translocations and homologous recombination, especially when cells with the CHO-Dhfr expression system are treated with methotrexate hydrate. The present method used in the industry for testing clones for their long-term stability of recombinant protein production is empirical, and it involves their cultivation over extended periods of time prior to the selection of the most suitable clone for further bioprocess development. The aim of the present study was the identification of marker genes that can predict stable expression of recombinant genes in particular clones early in the development stage.
Results
The transcriptome profiles of CHO clones with stable and unstable recombinant protein production were investigated over 10-weeks of cultivation, using a DNA microarray. We identified 14 genes that were differentially expressed between the stable and unstable clones already at 2 weeks from the beginning of the cultivation. Their expression was validated by reverse-transcription quantitative real-time PCR (RT-qPCR). Furthermore, the k-nearest neighbour algorithm approach shows that the combination of the gene expression patterns of only five of these 14 genes is sufficient to predict stable recombinant protein production in clones in the early phases of cell-line development.
Conclusions
The exact molecular mechanisms that cause unstable recombinant protein production are not fully understood. However, the expression profiles of some genes in clones with stable and unstable recombinant protein production allow prediction of such instability early in the cell-line development stage. We have thus developed a proof-of-concept for a novel approach to eliminate unstable clones in the CHO-Dhfr expression system, which saves time and labour-intensive work in cell-line development.
Ključne besede: CHO cell line, stable recombinant protein production, gene expression, RT-qPCR, DNA microarray, mMarker genes
Objavljeno v DiRROS: 29.07.2024; Ogledov: 372; Prenosov: 348
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1106. Recent ostracods (Crustacea: Ostracoda) of Alpine springs and adjacent springbrooks of the Southern Limestone Alps, SloveniaNataša Mori, 2015, izvirni znanstveni članek Povzetek: The ecology and distribution of ostracods in Alpine springs and springbrooks from Slovenia is presented. Benthos sampling was performed and major environmental characteristics (discharge, substrate composition, temperature, dissolved oxygen, conductivity, pH, alkalinity, sulphate, nitrate, calcium, magnesium) were measured in 12 springs and adjacent springbrooks. Sampling campaign was carried out on six sampling occasions (spring, summer, autumn in 2009 and 2010). Fourteen ostracod species were found among other fauna. The commonest and most abundant species were Psychrodromus fontinalis (Wolf, 1920) and Cavernocypris subterranea (Wolf, 1920), while the other species occurred at one or two sites at the most. Substrate composition and water temperature were statistically significant variables in explaining ostracod assemblages composition in this study.
Ključne besede: microcrustacea, species-environment relationship, biodiversity, distribution
Objavljeno v DiRROS: 29.07.2024; Ogledov: 344; Prenosov: 222
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1107. Heterogeneous glioblastoma cell cross-talk promotes phenotype alterations and enhanced drug resistanceAna Koren, Helena Motaln, Živa Ramšak, Kristina Gruden, Christian Schichor, Tamara Lah Turnšek, 2015, izvirni znanstveni članek Povzetek: Glioblastoma multiforme is the most lethal of brain cancer, and it comprises a heterogeneous mixture of functionally distinct cancer cells that affect tumor progression. We examined the U87, U251, and U373 malignant cell lines as in vitro models to determine the impact of cellular cross-talk on their phenotypic alterations in co-cultures. These cells were also studied at the transcriptome level, to define the mechanisms of their observed mutually affected genomic stability, proliferation, invasion and resistance to temozolomide. This is the first direct demonstration of the neural and mesenchymal molecular fingerprints of U87 and U373 cells, respectively. U87-cell conditioned medium lowered the genomic stability of U373 (U251) cells, without affecting cell proliferation. In contrast, upon exposure of U87 cells to U373 (U251) conditioned medium, U87 cells showed increased genomic stability, decreased proliferation rates and increased invasion, due to a plethora of produced cytokines identified in the co-culture media. This cross talk altered the expression 264 genes in U87 cells that are associated with proliferation, inflammation, migration, and adhesion, and 221 genes in U373 cells that are associated with apoptosis, the cell cycle, cell differentiation and migration. Indirect and direct co-culturing of U87 and U373 cells showed mutually opposite effects on temozolomide resistance. In conclusion, definition of transcriptional alterations of distinct glioblastoma cells upon co-culturing provides better understanding of the mechanisms of glioblastoma heterogeneity, which will provide the basis for more informed glioma treatment in the future.
Ključne besede: glioblastoma heterogeneity, U87 cells, temozolomide resistance, cellular cross-talk, transcriptomics
Objavljeno v DiRROS: 29.07.2024; Ogledov: 360; Prenosov: 260
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1108. Metabolic potential, respiration rate and their relationship in offspring of different sizes of marble trout (Salmo marmoratus Cuvier)Anton Brancelj, Tatjana Simčič, Dušan Jesenšek, 2015, izvirni znanstveni članek Povzetek: The size and composition of fish eggs are related to female’s characteristics, such as age, size and individual conditions,
and they have an impact on the properties of offspring that are important for their fitness. Electron transport system (ETS) activity and respiration rate (R) of early life history stages (i.e. non-fertilized eggs, eggs at eyed stage and larvae with yolk sac) of 13 females of marble trout (Salmo marmoratus) were measured separately in order to determine their metabolic
properties in relation to size. The results showed that larger females produced larger eggs in higher numbers. Growth experiments on the survival of offspring of a single female revealed that the survival rate of early embryos was higher for
smaller eggs during the earliest stages, but ultimately the percentage of surviving larvae did not correlate with egg size. The ETS activities and respiration rates of non-fertilized eggs, eyed eggs and larvae differed significantly between 13 females. Both parameters increased with increasing dry mass of the early life history stages, but the increase of respiration rate was greater than that of ETS activity. The lower ETS/R ratios in larger individuals therefore indicate that their energy metabolism was less adaptable to environmental changes than that of smaller ones. Larger egg size could be an advantage under favourable conditions, whereas smaller size could be optimal under stressing circumstances in which the higher metabolic potential enables production of the energy required for metabolism. This is first report on the
relationship between ETS activity and respiration rate of the early life history stages in salmonids.
Ključne besede: Salmo marmoratus, fitness, metabolism, ETS activity, ETS/R ratio, fishes
Objavljeno v DiRROS: 29.07.2024; Ogledov: 339; Prenosov: 196
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1109. Obročkanje ptic v Sloveniji leta 2014 in rezultati prvega telemetrijskega spremljanja selitvene poti afriške selivkeAl Vrezec, Dare Fekonja, Katarina Denac, 2015, izvirni znanstveni članek Povzetek: In 2014, 162 bird species were recorded during the bird ringing activities in Slovenia. Of 155 species, 62,275 birds were ringed, and 107 recoveries of birds ringed in Slovenia and found abroad, 148 foreign recoveries in Slovenia and 1395 local recoveries were recorded. The most frequently ringed species were Blackcap Sylvia atricapilla and Great Tit Parus major. As far as ringed nestlings are concerned, Great Tits and Barn Swalllows Hirundo rustica predominated. Considering the recoveries ringed of found birds abroad, the commonest were Black-headed Gulls Chroicocephalus ridibundus and Mute Swans Cygnus olor. The farthest recovery was a Barn Swallow Hirundo rustica found in the Democratic Republic of Congo (5171 km away). Among the more interesting finds was also the so far southernmost recovery of a Sand Martin Riparia riparia found in Israel. Let us also mention the first recovery of a Corncrake Crex crex, which bred and was ringed in 2013 at Planinsko polje (central Slovenia) and was found in the 2014 breeding season in the Czech Republic. Among rare species, two Little Buntings Emberiza pusilla were caught and ringed. After nine years, the Roller Coracias garrulus bred again in Slovenia in 2014 and its nestlings were ringed. The paper also brings the description of the migration route of the first African migrant, the Black Stork Ciconia nigra, marked with a GPS/GSM telemetric device, which migrated across the Adriatic Sea, Sicily and Sahara to Nigeria.
Ključne besede: obročkanje, telemetrija, črna štorklja, najdbe (biologija)
Objavljeno v DiRROS: 29.07.2024; Ogledov: 345; Prenosov: 239
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1110. Roles of the crotonyl-CoA carboxylase/reductase homologues in acetate assimilation and biosynthesis of immunosuppressant FK506 in Streptomyces tsukubaensisGregor Kosec, Hrvoje Petković, Špela Baebler, Kristina Gruden, Marko Blažič, 2015, izvirni znanstveni članek Povzetek: Background
In microorganisms lacking a functional glyoxylate cycle, acetate can be assimilated by alternative pathways of carbon metabolism such as the ethylmalonyl-CoA (EMC) pathway. Among the enzymes converting CoA-esters of the EMC pathway, there is a unique carboxylase that reductively carboxylates crotonyl-CoA, crotonyl-CoA carboxylase/reductase (Ccr). In addition to the EMC pathway, gene homologues of ccr can be found in secondary metabolite gene clusters that are involved in the provision of structurally diverse extender units used in the biosynthesis of polyketide natural products. The roles of multiple ccr homologues in the same genome and their potential interactions in primary and secondary metabolic pathways are poorly understood.
Results
In the genome of S. tsukubaensis we have identified two ccr homologues; ccr1 is located in the putative ethylmalonyl-CoA (emc) operon and allR is located on the left fringe of the FK506 cluster. AllR provides an unusual extender unit allylmalonyl-CoA (ALL) for the biosynthesis of FK506 and potentially also ethylmalonyl-CoA for the related compound FK520. We have demonstrated that in S. tsukubaensis the ccr1 gene does not have a significant role in the biosynthesis of FK506 or FK520 when cultivated on carbohydrate-based media. However, when overexpressed under the control of a strong constitutive promoter, ccr1 can take part in the biosynthesis of ethylmalonyl-CoA and thereby FK520, but not FK506. In contrast, if ccr1 is inactivated, allR is not able to sustain a functional ethylmalonyl-CoA pathway (EMC) and cannot support growth on acetate as the sole carbon source, even when constitutively expressed in the chimeric emc operon. This is somewhat surprising considering that the same chimeric emc operon results in production of FK506 as well as FK520, consistent with the previously proposed relaxed specificity of AllR for C4 and C5 substrates.
Conclusions
Different regulation of the expression of both ccr genes, ccr1 and allR, and their corresponding pathways EMC and ALL, respectively, in combination with the different enzymatic properties of the Ccr1 and AllR enzymes, determine an almost exclusive role of ccr1 in the EMC pathway in S. tsukubaensis, and an exclusive role of allR in the biosynthesis of FK506/FK520, thus separating the functional roles of these two genes between the primary and secondary metabolic pathways.
Ključne besede: streptomicete, Streptomyces tsukubaensis, sekundarni metaboliti, takrolimus, FK506, biosinteze poliketidov
Objavljeno v DiRROS: 29.07.2024; Ogledov: 342; Prenosov: 230
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