Die Retrotransposonen sind ubiquitäre Komponenten des eukaryotischen Genoms. In der Hefe Yarrowia lipolytica existiert neben anderen Retrotransposonen das Retrotransposon Ylt1 (Yarrowia lipolytica retrotransposon). Dieses Retrotransposon besteht aus den flankierenden "Long Terminal Repeat" (LTR), zeta von jeweils 714 Nukleotiden und interner Region, eta von 8025 Nukleotiden. Deswegen baut die gesamte Sequenz von 9453 Nukleotiden. Der etwa Bereich enthält einen durchgängigen offenen Leserahm (ORF) (826 bis 8687 Nukleotiden, 2621 Aminosäure). Die LTR Sequenzen enthalten nicht nur TATA-Box, sondern auch ein mögliches Terminations-Signal der Transkription (TAGT) sowie ein Polyadenylierungssignal (AATAAA). Eine Primer Bindungsstelle und ein Polypurin-Bereich sind ebenfalls innerhalb der Ylt1 Sequenz enthalten. Dieses Retrotransposon enthält codierenden Bereiche für ein Gag-Strukturprotein (Gag), Protease (PR), Reverse Transkriptase (RT), RNase H (RH) und Integrase (IN). Das Retrotransposons Ylt1 kommt in verschiedenen Stämmen der Hefe Y. lipolytica vor. Die Transpositionsaktivität dieses Retrotransposon war nachweisbar. Die Aktivität des LTR-Promotors ist von der Kohlenstoffquellen abhängig. Acetat und Ethanol bewirken eine Erhöhung der Promotoraktivität auf das 4fache der basalen Aktivität. Nachdem der Ylt1-ORF unter Kontrolle des ICL1-Promotors exprimiert wurde, konnten jeweils etwa 140 kDa (mit dem Anti HA-Antikörper) bzw. ca. 74 kDa (mit Anti GFP-Antikörper) große Proteine aus den zwei DNA Konstrukte nachgewiesen werden. / The retrotransposons are ubiquitous components of eukaryotic genome. The retrotransposon Ylt1 (Yarrowia lipolytica retrotransposon) was detected in the genome of the dimorphic yeast Yarrowia lipolytica. This retrotransposon is 9453 nucleotides in length and contains two identical long terminal repeats (LTR), zeta of 714 nucleotides and internal region, eta of 8025 nucleotides. The LTR sequences contain not only TATA-Box but also predicted termination signal for transcription (TATG) and polyadenylation signal (AATAAA). The both putative primer binding site (PBS) and polypurine tract were found in the retrotransposon Ylt1 sequence. This retrotransposon contains a single open reading frame (ORF) (826 - 8687 nucleotides, 2621 amino acids), which encodes Gag protein (Gag), protease (PR), reverse transcriptase (RT), RNase H (RH) and integrase (IN). The distribution of retrotransposon Ylt1 among Y. lipolytica strains indicated that the full length elements as well as solo LTR are abundant in several strains. The transposition activity of retrotransposon Ylt1 on acetate as a carbon source was also observed. The promoter activity of the LTR sequence depends on the carbon source. Acetate and ethanol activated 4 fold from the basal activity of the LTR promoter. After expression of Ylt1-ORF under the strong inducible ICL1 promoter from two DNA constructs, which contain HA or GFP epitope, a protein about 140 kDa (with anti HA-antibody) or 75 kDa (with anti GFP-antibody) were detected.

Die Retrotransposonen sind ubiquitäre Komponenten des eukaryotischen Genoms. In der Hefe Yarrowia lipolytica existiert neben anderen Retrotransposonen das Retrotransposon Ylt1 (Yarrowia lipolytica retrotransposon). Dieses Retrotransposon besteht aus den flankierenden "Long Terminal Repeat" (LTR), zeta von jeweils 714 Nukleotiden und interner Region, eta von 8025 Nukleotiden. Deswegen baut die gesamte Sequenz von 9453 Nukleotiden. Der etwa Bereich enthält einen durchgängigen offenen Leserahm (ORF) (826 bis 8687 Nukleotiden, 2621 Aminosäure). Die LTR Sequenzen enthalten nicht nur TATA-Box, sondern auch ein mögliches Terminations-Signal der Transkription (TAGT) sowie ein Polyadenylierungssignal (AATAAA). Eine Primer Bindungsstelle und ein Polypurin-Bereich sind ebenfalls innerhalb der Ylt1 Sequenz enthalten. Dieses Retrotransposon enthält codierenden Bereiche für ein Gag-Strukturprotein (Gag), Protease (PR), Reverse Transkriptase (RT), RNase H (RH) und Integrase (IN). Das Retrotransposons Ylt1 kommt in verschiedenen Stämmen der Hefe Y. lipolytica vor. Die Transpositionsaktivität dieses Retrotransposon war nachweisbar. Die Aktivität des LTR-Promotors ist von der Kohlenstoffquellen abhängig. Acetat und Ethanol bewirken eine Erhöhung der Promotoraktivität auf das 4fache der basalen Aktivität. Nachdem der Ylt1-ORF unter Kontrolle des ICL1-Promotors exprimiert wurde, konnten jeweils etwa 140 kDa (mit dem Anti HA-Antikörper) bzw. ca. 74 kDa (mit Anti GFP-Antikörper) große Proteine aus den zwei DNA Konstrukte nachgewiesen werden. / The retrotransposons are ubiquitous components of eukaryotic genome. The retrotransposon Ylt1 (Yarrowia lipolytica retrotransposon) was detected in the genome of the dimorphic yeast Yarrowia lipolytica. This retrotransposon is 9453 nucleotides in length and contains two identical long terminal repeats (LTR), zeta of 714 nucleotides and internal region, eta of 8025 nucleotides. The LTR sequences contain not only TATA-Box but also predicted termination signal for transcription (TATG) and polyadenylation signal (AATAAA). The both putative primer binding site (PBS) and polypurine tract were found in the retrotransposon Ylt1 sequence. This retrotransposon contains a single open reading frame (ORF) (826 - 8687 nucleotides, 2621 amino acids), which encodes Gag protein (Gag), protease (PR), reverse transcriptase (RT), RNase H (RH) and integrase (IN). The distribution of retrotransposon Ylt1 among Y. lipolytica strains indicated that the full length elements as well as solo LTR are abundant in several strains. The transposition activity of retrotransposon Ylt1 on acetate as a carbon source was also observed. The promoter activity of the LTR sequence depends on the carbon source. Acetate and ethanol activated 4 fold from the basal activity of the LTR promoter. After expression of Ylt1-ORF under the strong inducible ICL1 promoter from two DNA constructs, which contain HA or GFP epitope, a protein about 140 kDa (with anti HA-antibody) or 75 kDa (with anti GFP-antibody) were detected.

The retrotransposon Ylt1 was described previously from the genome of the dimorphic fungus Yarrowia lipolytica. Remarkably, Ylt1 is currently the largest LTR retrotransposon reported from fungal genomes. However, little was known about its biology and its interactions with host genome. So, the aim of this work was the characterization of properties of Ylt1.Analysis of proteins encoded by Ylt1 (Gag protein and integrase) was carried out during this work. To enable their detection, both proteins were tagged with HA epitopes. The sizes of Gag protein and putative precursors of Gag protein and integrase were estimated, and a model for the proteolytic processing of the polyprotein of Ylt1 was proposed. It was shown that Gag protein of Ylt1 is about 2-fold larger than Gag proteins of other studied yeast retrotransposons. An analysis of Ylt1 expression was also performed. Production of the Ylt1 Gag protein under different conditions was analyzed by Western blotting. Expression of Ylt1 occurred on all tested carbon sources. The amount of Ylt1 decreased rapidly upon transition to stationary growth phase, in the presence of copper sulfate and under heat shock conditions. It is suggested that Ylt1 is expressed in actively growing cells, whereas stress conditions have a negative impact on its expression. Such expression pattern was not previously reported for other yeast retrotransposons. Activity of Ylt1 in vivo was characterized using an Ylt1 elements tagged with SUC2 gene of Saccharomyces cerevisiae. Mobilization of the marked Ylt1 element and its transposition from autonomous plasmid into host genome was observed in performed experiments. Obtained results strongly support the idea that Ylt1 is transpositionally active. Formation of tandem repeats by newly inserted Ylt1 elements was observed in several cases. It is suggested that integrase function was affected in this case, and that the integration was mediated by homologous recombination instead. Analysis of the Ylt1 insertion specificity and of the Ylt1 distribution in the genome of Y. lipolytica E150 was done. The remarkable sequence specificity of Ylt1 insertions, which is unusual for LTR retrotransposons, was revealed during this analysis. Also, it was shown that Ylt1 insertions are found mainly in intergenic regions, often at a significant distance (>500 bp) from the next reading frame. No association of Ylt1 insertions with tRNA genes was observed. Searches for Ylt1-related elements in the Y. lipolytica genome database were performed. The novel Ty3/gypsy element Tyl6 was found in the genome of Y. lipolytica E150. The sequence analysis of this element was carried out. It was shown that structural properties of Tyl6 resemble the properties of the Ty3 element of S. cerevisiae. However, two reading frames of Tyl6 (gag and pol) are separated by -1 frame-shift, which was not previously reported for retrotransposons of hemiascomycetous yeasts. Phylogenetic analysis placed Tyl6 within chromoviruses, and the Tse3 element of S. exiguus was shown to be the closest relative of Tyl6. The distribution of Tyl6 among Y. lipolytica strains was analyzed. Interestingly, the novel element was found only in strains derived from the strain YB423-12. The strains of independent origin included in the analysis were shown to be Tyl6-free. The same distribution was previously reported for the retrotransposon Ylt1 and for the DNA transposon Mutyl. Two models of the evolution of transposable elements in Y. lipolytica genome were proposed based on these results.

The retrotransposon Ylt1 was described previously from the genome of the dimorphic fungus Yarrowia lipolytica. Remarkably, Ylt1 is currently the largest LTR retrotransposon reported from fungal genomes. However, little was known about its biology and its interactions with host genome. So, the aim of this work was the characterization of properties of Ylt1.Analysis of proteins encoded by Ylt1 (Gag protein and integrase) was carried out during this work. To enable their detection, both proteins were tagged with HA epitopes. The sizes of Gag protein and putative precursors of Gag protein and integrase were estimated, and a model for the proteolytic processing of the polyprotein of Ylt1 was proposed. It was shown that Gag protein of Ylt1 is about 2-fold larger than Gag proteins of other studied yeast retrotransposons. An analysis of Ylt1 expression was also performed. Production of the Ylt1 Gag protein under different conditions was analyzed by Western blotting. Expression of Ylt1 occurred on all tested carbon sources. The amount of Ylt1 decreased rapidly upon transition to stationary growth phase, in the presence of copper sulfate and under heat shock conditions. It is suggested that Ylt1 is expressed in actively growing cells, whereas stress conditions have a negative impact on its expression. Such expression pattern was not previously reported for other yeast retrotransposons. Activity of Ylt1 in vivo was characterized using an Ylt1 elements tagged with SUC2 gene of Saccharomyces cerevisiae. Mobilization of the marked Ylt1 element and its transposition from autonomous plasmid into host genome was observed in performed experiments. Obtained results strongly support the idea that Ylt1 is transpositionally active. Formation of tandem repeats by newly inserted Ylt1 elements was observed in several cases. It is suggested that integrase function was affected in this case, and that the integration was mediated by homologous recombination instead. Analysis of the Ylt1 insertion specificity and of the Ylt1 distribution in the genome of Y. lipolytica E150 was done. The remarkable sequence specificity of Ylt1 insertions, which is unusual for LTR retrotransposons, was revealed during this analysis. Also, it was shown that Ylt1 insertions are found mainly in intergenic regions, often at a significant distance (>500 bp) from the next reading frame. No association of Ylt1 insertions with tRNA genes was observed. Searches for Ylt1-related elements in the Y. lipolytica genome database were performed. The novel Ty3/gypsy element Tyl6 was found in the genome of Y. lipolytica E150. The sequence analysis of this element was carried out. It was shown that structural properties of Tyl6 resemble the properties of the Ty3 element of S. cerevisiae. However, two reading frames of Tyl6 (gag and pol) are separated by -1 frame-shift, which was not previously reported for retrotransposons of hemiascomycetous yeasts. Phylogenetic analysis placed Tyl6 within chromoviruses, and the Tse3 element of S. exiguus was shown to be the closest relative of Tyl6. The distribution of Tyl6 among Y. lipolytica strains was analyzed. Interestingly, the novel element was found only in strains derived from the strain YB423-12. The strains of independent origin included in the analysis were shown to be Tyl6-free. The same distribution was previously reported for the retrotransposon Ylt1 and for the DNA transposon Mutyl. Two models of the evolution of transposable elements in Y. lipolytica genome were proposed based on these results.