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3.
Mol Genet Genomics ; 267(6): 829-34, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12207231

ABSTRACT

I factors are non-LTR retrotransposons of Drosophila melanogaster that transpose at high frequency in the germline of females resulting from appropriate crosses, allowing in vivo studies of the retrotransposition process. Reverse transcription of a full-length RNA intermediate is thought to occur at the site of integration, using a 3' hydroxyl group generated by endonucleolytic cleavage of the genomic DNA to prime synthesis of the first cDNA strand. This target-primed reverse transcription (TPRT) process is mediated by endonuclease and reverse transcriptase activities encoded by the element. We have designed a molecularly tagged, endonuclease-defective I element that can be mobilised with high efficiency by constructs that express the product of the I factor ORF2 in trans. This indicates that the endonuclease activity required for retrotransposition of the I factor can be provided in trans. Using this system, we show that the endonuclease domain of the R1Bm retrotransposon from Bombyx mori cannot functionally replace that of the I factor.


Subject(s)
Drosophila melanogaster/genetics , Genetic Complementation Test , Retroelements , Animals , Animals, Genetically Modified , Crosses, Genetic , Endonucleases/metabolism , Female , Male , Sequence Analysis, DNA
4.
Oncogene ; 18(46): 6222-32, 1999 Nov 04.
Article in English | MEDLINE | ID: mdl-10597220

ABSTRACT

Cell cycle modulation of cyclin A expression is due to the periodic relief of a transcriptional repression mediated by a bipartite negative DNA regulatory region. The 5' element (Cell Cycle Responsive Element: CCRE; cell Cycle Dependent Element: CDE) is clearly occupied in a cyclic manner in vivo, whereas the 3' element, whose sequence is shared by B-myb, cdc25C and cdc2 genes (cell Cycle gene Homology Region: CHR), is involved in more subtle interactions. Mutation of either element results in complete deregulation of cyclin A promoter activity. Whereas some reports claim that E2F/DP can bind to the CCRE/CDE, the nature of the protein(s) interacting with the CHR is unknown. In the present work we have characterized an activity present in quiescent cells and absent in cells blocked in S phase, which binds specifically to cyclin A CHR, but not to B-myb, or to cdc25C, or to cdc2 CHRs. A 90 kD protein, named CHF (cyclin A CHR binding factor), has been identified through preparative electrophoresis and UV crosslinking experiments. In order to address in more functional terms the binding of CHF to cyclin A CHR, we developed in vitro and in vivo oligonucleotide competition assays. Both in vitro transcription and in vivo microinjection experiments demonstrate that a functional difference exists between the composite CCRE/CDE-CHR repressor regions of cell cycle regulated genes such as cyclin A and cdc25C.


Subject(s)
Cell Cycle/genetics , Cyclin A/genetics , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , T-Lymphocytes/metabolism , Transcription Factors/isolation & purification , Transcription, Genetic , Animals , Base Sequence , Binding Sites , Binding, Competitive , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Humans , Hydroxyurea/pharmacology , Macromolecular Substances , Mice , Microinjections , Molecular Sequence Data , Molecular Weight , Resting Phase, Cell Cycle , S Phase/drug effects , T-Lymphocytes/drug effects , Transcription Factors/metabolism
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