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      Gag proteins of the two Drosophila telomeric retrotransposons are targeted to chromosome ends

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          Abstract

          Drosophila telomeres are formed by two non-LTR retrotransposons, HeT-A and TART, which transpose only to chromosome ends. Successive transpositions of these telomeric elements yield arrays that are functionally equivalent to the arrays generated by telomerase in other organisms. In contrast, other Drosophila non-LTR retrotransposons transpose widely through gene-rich regions, but not to ends. The two telomeric elements encode very similar Gag proteins, suggesting that Gag may be involved in their unique targeting to chromosome ends. To test the intrinsic potential of these Gag proteins for targeting, we tagged the coding sequences with sequence of GFP and expressed the constructs in transiently transfected Drosophila-cultured cells. Gag proteins from both elements are efficiently transported into the nucleus where the protein from one element, HeT-A, forms structures associated with chromosome ends in interphase nuclei. Gag from the second element, TART, moves into telomere-associated structures only when coexpressed with HeT-A Gag. The results suggest that these Gag proteins are capable of delivering the retrotransposons to telomeres, although TART requires assistance from HeT-A. They also imply a symbiotic relationship between the two elements, with HeT-A Gag directing the telomere-specific targeting of the elements, whereas TART provides reverse transcriptase for transposition.

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          Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition.

          R2 is a non-LTR retrotransposable element that inserts at a specific site in the 28S rRNA genes of most insects. We have expressed the open reading frame of the R2 element from Bombyx mori, R2Bm, in E. coli and shown that it encodes both sequence-specific endonuclease and reverse transcriptase activities. The R2 protein makes a specific nick in one of the DNA strands at the insertion site and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of its RNA transcript. After reverse transcription, cleavage of the second DNA strand occurs. A similar mechanism of insertion may be used by other non-LTR retrotransposable elements as well as short interspersed nucleotide elements.
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            The age and evolution of non-LTR retrotransposable elements.

            A comprehensive phylogenetic analysis was conducted of non-long-terminal-repeat (non-LTR) retrotransposons based on an extended sequence alignment of their reverse transcriptase (RT) domain. The 440 amino acid positions used included a region proposed to be similar to the "thumb" of the right-handed RT structure found in retroviruses. All identified non-LTR elements could be grouped into 11 distinct clades. Using the rates of sequence change derived from studies of the vertical inheritance of R1 and R2 elements in arthropods as a comparison, we found no evidence for the horizontal transmission of non-LTR elements. Assuming vertical descent, the phylogeny suggested that non-LTR elements are as old as eukaryotes, with each of the 11 clades dating back to the Precambrian era. The analysis enabled us to propose a simple chronology for the acquisition of different enzymatic domains in the evolution of the non-LTR class of retrotransposons. The first non-LTR elements were sequence specific by virtue of a restriction-enzyme-like endonuclease located downstream of the RT domain. Evolving from this original group were elements (eight clades) that acquired an apurinic-apyrimidic endonuclease-like domain upstream of the RT domain. Finally, four of these clades have inherited an RNase H domain downstream of the RT domain. The phylogenies of the AP endonuclease and RNase H domains were also determined for this report and are consistent with the monophyletic acquisition of these domains. These studies represent the most comprehensive effort to date to trace the evolution of a major class of transposable elements.
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              Displacement of sequence-specific transcription factors from mitotic chromatin.

              The general inhibition in transcriptional activity during mitosis abolishes the stress-inducible expression of the human hsp70 gene. Among the four transcription factors that bind to the human hsp70 promoter, the DNA-binding activities of three (C/EBP, GBP, and HSF1) were normal, while Sp1 showed reduced binding activity in mitotic cell extracts. In vivo footprinting and immunocytochemical analyses revealed that all of the sequence-specific transcription factors were displaced from promoter sequences as well as from bulk chromatin during mitosis. The correlation of transcription factor displacement with chromatin condensation suggests an involvement of chromatin structure in mitotic repression. However, retention of DNase I hypersensitivity suggests that the hsp70 promoter was not organized in a canonical nucleosome structure in mitotic chromatin. Displacement of transcription factors from mitotic chromosomes could present another window in the cell cycle for resetting transcriptional programs.
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                Author and article information

                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                11 November 2002
                : 159
                : 3
                : 397-402
                Affiliations
                [1 ]Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
                [2 ]School of Biological Sciences, University of Kentucky, Lexington, KY 40506
                Author notes

                Address correspondence to Mary-Lou Pardue, Dept. of Biology, 68-670, Massachusetts Institute of Technology, Cambridge, MA 02139. Tel.: (617) 253-6741. Fax: (617) 253-8699. E-mail: mlpardue@ 123456mit.edu

                Article
                200205039
                10.1083/jcb.200205039
                2173066
                12417578
                e26bb595-5b1a-47e2-befa-5763def7523e
                Copyright © 2002, The Rockefeller University Press
                History
                : 6 May 2002
                : 5 September 2002
                : 9 September 2002
                Categories
                Report

                Cell biology
                intracellular targeting; het-a; tart; retrovirus; telomere
                Cell biology
                intracellular targeting; het-a; tart; retrovirus; telomere

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