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Seabuckthorn (Hippophae rhamnoides) is a dioecious shrub commonly used in the pharmaceutical, cosmetic, and environmental industry as a source of oil, minerals and vitamins. In this study, we analyzed the transposable elements and satellites in its genome.We carried out Illumina DNA sequencing and reconstructed the main repetitive DNA sequences. For data analysis, we developed a new bioinformatics approach for advanced satellite DNA analysis and showed that about 25% of the genome consists of satellite DNA and about 24% is formed of transposable elements, dominated by Ty3/Gypsy and Ty1/Copia LTR retrotransposons. FISH mapping revealed X chromosome-accumulated, Y chromosome-specific or both sex chromosomes-accumulated satellites but most satellites were found on autosomes. Transposable elements were located mostly in the subtelomeres of all chromosomes. The 5S rDNA and 45S rDNA were localized on one autosomal locus each. Although we demonstrated the small size of the Y chromosome of the seabuckthorn and accumulated satellite DNA there, we were unable to estimate the age and extent of the Y chromosome degeneration. Analysis of dioecious relatives such as Shepherdia would shed more light on the evolution of these sex chromosomes., Rakytník (Hippophae rhamnoides) je dvoudomý keř bežně používán ve farmaceutickém, kosmetickém a enviromentálním průmyslu jako zdroj oleje, minerálů a vitamínů. V této studii jsme analyzovali transpozony a satelity v jeho genomu. Vykonali jsme Illumina sekvenování a rekonstruovali jsme hlavní repetitivní DNA sekvence. Pro analýzu dat jsme vyvinuli nový bioinformatický přístup pro pokročilou analýzu satelitní DNA a ukázali jsme, že okolo 25% genomu tvoří satelitní DNA a asi 24% je tvořených transpozony, u kterých dominují Ty3/Gypsy a Ty1/Copia LTR retrotranspozony. FISH mapování odhalilo satelit akumulovaný na X chromozomu, Y specifický satelit a satelit akumulovaný na obou pohlavních chromozomech, přičemž většina satelitů byla nalzena na autosomech. Transpozibilní elementy byly umístněny většinou v subtelomerách všech chromozomů. 5S rDNA a 45S rDNA byly lokalizovány každá na jednom autosomálním lokusu. Přestože jsme demonstrovali malou velikost Y chromozomu a na něm akumulovanou satelitní DNA, nebyli jsme schopni odhadnou věk a rozsah degenerace Y chromozomu. Analýza dvoudomých příbuzných jako je např. Shepherdia by lépe objasnila evoluci pohlavních chromosomů.

Genomic DNA of Crypthecodinium cohnii has been extracted in the presence of cetylmethylammonium bromide and hydrolysed by 13 restriction enzymes. No typical ladder-like pattern or isolated band of satellite sequences were found with any of these enzymes. A “mini” genomic DNA library had been made and screened by reverse hybridization to isolate highly repeated sequences. Seven such DNA fragments were sequenced. The copy number of one of them (Cc18), 226 bp long, was estimated at around 25,000, representing 0.06% of the total genome. Cc18 was found to be included in a higher fragment of 3.0 kb by Southern blot analysis after cleavage by PstI. This higher molecular weight fragment could be composed either of tandemly repeated Cc18 sequences, or by only one or a very low copy number of Cc18. In this latter case, these fragments, also repeated 25,000 times would represent 1 to 2% of the total genome. Genomic localization of Cc18 by in situ hybridization on squashed C. cohnii cells showed that it was widely distributed on the different chromosomes. All the chromosomes observed displayed Cc18 labeling, which appeared homogeneously distributed. The ability of Cc18 to be a specific molecular marker to distinguish sibling C. cohnii species is discussed.

Genomic DNA of Crypthecodinium cohnii has been extracted in the presence of cetylmethylammonium bromide and hydrolysed by 13 restriction enzymes. No typical ladder-like pattern or isolated band of satellite sequences were found with any of these enzymes. A “mini” genomic DNA library had been made and screened by reverse hybridization to isolate highly repeated sequences. Seven such DNA fragments were sequenced. The copy number of one of them (Cc18), 226 bp long, was estimated at around 25,000, representing 0.06% of the total genome. Cc18 was found to be included in a higher fragment of 3.0 kb by Southern blot analysis after cleavage by PstI. This higher molecular weight fragment could be composed either of tandemly repeated Cc18 sequences, or by only one or a very low copy number of Cc18. In this latter case, these fragments, also repeated 25,000 times would represent 1 to 2% of the total genome. Genomic localization of Cc18 by in situ hybridization on squashed C. cohnii cells showed that it was widely distributed on the different chromosomes. All the chromosomes observed displayed Cc18 labeling, which appeared homogeneously distributed. The ability of Cc18 to be a specific molecular marker to distinguish sibling C. cohnii species is discussed.

White (WAT) and brown (BAT) adipose tissue are tissues of energy storage and energy dissipation, respectively. Experimental evidence suggests that brown and white preadipocytes are differentially determined, but so far not much is known about the genetic control of this determination process. The aim of this study was to identify differentially expressed genes involved in brown and white preadipocyte development. Using representational difference analysis (cDNA RDA) and DNA microarray screening, we identified four genes with higher expression in white preadipocytes (three different complement factors and -6 fatty acid desaturase) and seven genes with higher expression levels in brown preadipocytes, of which three are structural genes implicated in cell adhesion and cytoskeleton organization (fibronectin, -actinin-4, metargidin) and four that might function in gene transcription and protein synthesis (vigilin, necdin, snRNP polypeptide A, and a homolog to human hepatocellular carcinoma-associated protein). The expression profile of these genes was analyzed during preadipocyte differentiation, upon ß-adrenergic stimulation, and in WAT and BAT tissue in vivo compared with references genes such as peroxisome proliferator-activated receptor- (PPAR), uncoupling protein 1 (UCP1), cytochrome c oxidase

We have previously shown that the immunosuppressant cyclosporine A (CsA) increases the activity, the protein level, and the steady-state levels of the mRNA of the endothelial nitric-oxide synthase (eNOS) gene in bovine aortic endothelial cells (BAEC). We have now investigated the mechanisms responsible for these effects. Preincubation with an inhibitor of RNA polymerase II abolished CsA-induced eNOS up-regulation. Nuclear run-on experiments demonstrated a 1.6-fold increase in the induction of eNOS gene by CsA. In agreement with these results, transient transfections showed that CsA augmented the transactivation of the eNOS promoter. Electrophoretic mobility shift assays showed an increase in the activator protein-1 (AP-1) DNA binding activity in BAEC treated with CsA. An increase in the level of c-fos mRNA and in the nuclear content of c-Fos protein was detected in BAEC treated with CsA. Site-directed mutagenesis of the AP-1 cis-regulatory element in the context of the human eNOS promoter resulted in the abrogation of the induction mediated by CsA. Hence, up-regulation ofeNOS mRNA by CsA is a transcriptional phenomenon involving the proximal AP-1 site in the 5′-regulatory region of the human eNOS gene. Furthermore, our data exemplify how immunosuppressive drugs may result in the regulation of specific genes involved in the homeostasis of endothelial function, such aseNOS. Whereas cyclosporine A (CsA)1 still stands as a cornerstone therapy for immunosuppression, its use encompasses serious side effects among which nephrotoxicity and hypertension are often encountered. The pathogenesis of their appearance has been investigated in clinical studies and animal models, but a complete understanding is still elusive. The partial reversibility of these effects suggests that the underlying disturbance has a functional component. Acute nephrotoxicity is associated with renal vasoconstriction and a reduced glomerular filtration rate. A significant amount of data support that th

Two full-length lipoxygenase cDNA sequences (LoxB and LoxC) from barley (Hordeum distichum cv. L. Triumph) are described. The cDNAs share high homology with the barley LoxA cDNA. Southern blotting experiments indicate single copy numbers of the three lipoxygenase genes. RFLP mapping revealed the presence of single lipoxygenase loci. LoxA and LoxB map on chromosome 4 and LoxC on chromosome 7. Two isoenzymes, LOX1 and LOX2, have been purified previously from germinating barley and characterized. LOX1 is encoded by LoxA, while LOX2 is encoded by LoxC. The product related to the third cDNA (loxB) has not been identified so far, suggesting a low protein abundance for the corresponding isoform in barley. Transcripts corresponding with these LOX genes are predominantly observed in grain and in seedling, whereas transcripts corresponding to LoxB and LoxC are also observed in mature vegetative tissue. No lipoxygenase mRNA could be detected in aleurone layer of germinating grain. No significant differences in lipoxygenase mRNA levels were observed in developing grains grown under dormant or non-dormant conditions, suggesting that LOX is not directly involved in induction of grain dormancy. Chemicals/CAS: Antibodies, Monoclonal; Isoenzymes; Lipoxygenase, EC 1.13.11.12; Recombinant Proteins; RNA, Messenger

Using fluorescence in situ hybridization (FISH) we localized introduced genes on metaphase chromosomes of barley, wheat, and triticale transformed by microprojectile bombardment of microspores and scutellar tissue with the pDB1 plasmid containing the uid A and bar genes. Thirteen integration sites were detected in the nine lines analysed. Southern analysis showed that three or more copies of the plasmid were present in the lines. In a triticale line containing four copies three different integration sites were identified, indicating that the method described is sensitive enough for the detection of single-copy integrations. There was a slight tendency towards the localization of transgenes in distal chromosome regions. Using the GAA-satellite sequence for chromosome banding, the chromosomes containing the inserted genes were identified in most cases. Two barley lines derived from the same transformant showed a totally different integration pattern. Southern analysis confirmed that the inserted genes were segregating independently, resulting in different integration patterns among the progeny lines. The application of the FISH technique for the analysis of transgenic plants is discussed.

Using fluorescence in situ hybridization (FISH) we localized introduced genes on metaphase chromosomes of barley, wheat, and triticale transformed by microprojectile bombardment of microspores and scutellar tissue with the pDB1 plasmid containing the uid A and bar genes. Thirteen integration sites were detected in the nine lines analysed. Southern analysis showed that three or more copies of the plasmid were present in the lines. In a triticale line containing four copies three different integration sites were identified, indicating that the method described is sensitive enough for the detection of single-copy integrations. There was a slight tendency towards the localization of transgenes in distal chromosome regions. Using the GAA-satellite sequence for chromosome banding, the chromosomes containing the inserted genes were identified in most cases. Two barley lines derived from the same transformant showed a totally different integration pattern. Southern analysis confirmed that the inserted genes were segregating independently, resulting in different integration patterns among the progeny lines. The application of the FISH technique for the analysis of transgenic plants is discussed.