Nucleotide sequence analysis and enhancer function of long terminal repeats associated with an endogenous African green monkey retroviral DNA.

The nucleotide sequence and enhancer activity of the long terminal repeats (LTRs) associated with a cloned endogenous African green monkey (AGM) retroviral DNA designated as lambda-AGM-1 was studied. A unique feature of the endogenous AGM proviral LTRs was the presence of multiple copies of two types of directly repeating units in the U3 region: 16 8-base-pair (bp) repeats were present in the 5' LTR and 12 were present in the 3' LTR which were bound by a 6-bp perfect direct repeat; tandem duplication of a 32-bp sequence resulted in 3.5 copies in the 5' LTR and 2.5 copies in the 3' LTR. Nucleotide sequence homology was seen between the 8-bp direct repeats located in the AGM proviral LTRs and a 10-bp repeat unit of the deca-satellite present in AGM cellular DNA. The 32-bp repeats of the AGM proviral LTRs contained sequences which were related to the SV40 21-bp repeats and to the "core" of the SV40 72-bp enhancer element. Furthermore, the AGM provirus was distinct from known infectious retroviruses due to the presence of a primer-binding sequence complementary to the 3' terminus of mammalian tRNAGly. Functional analysis of the 3' LTR present in lambda-AGM-1 DNA by chloramphenicol acetyltransferase assay demonstrated enhancer activity associated with the 32-bp direct repeats. Sequences outside the 32-bp unit were necessary for full activator function, suggesting the presence of multiple enhancer domains in the AGM provirus.     

Organization and distribution pattern of MGLR-3, a novel retrotransposon in the rice blast fungus Magnaporthe grisea

A specific telomere was deleted in spontaneous, gain-of-virulence mutants derived from a rice pathogen of Magnaporthe grisea. Three different types of transposons, including Pot2, Mg-SINE, and a novel, 6-kb-long LTR (long terminal repeat)-type retrotransposon designated MGLR-3, were identified on this chromosomal end. The 114-bp-long telomeric repeat is immediately followed by the 3' LTR of MGLR-3. A truncated copy of Pot2 immediately flanks the 5' LTR, suggesting that this telomere was generated by a transposition event of MGLR-3 into this Pot2 element, causing the breakage of a chromosome. The subsequent addition of a telomeric repeat to the 3' LTR of MGLR-3 most probably repaired the broken end of the chromosome. Mg-SINE is located 25 bp away from the truncated Pot2 element. MGLR-3 exhibited strong homology to various gypsy-class retrotransposons, including grh and MAGGY in M. grisea. MGLR-3 is ubiquitous regardless of the host of origin.     

Two subfamilies of murine retrotransposon ETn sequences

Early transposon (ETn) elements are 5.7-kb retrotransposons found in the murine genome. We have sequenced large portions of two ETn elements that have apparently transposed within the DNA of a murine myeloma cell line, P3.26Bu4. One of the transposed ETn elements has 5' and 3' long terminal repeats (LTRs) that are exact duplicates of each other and has a 6-bp target site duplication. These results suggest that this element, which inserted into an immunoglobulin gamma 1 switch region, moved by a retrotransposition process. Our nucleotide sequences confirm that individual ETn elements are very similar to one another and lack open reading frames. However, the ETn sequences reported here and those previously described differ significantly near their 5' LTRs, including 200 bp of weak similarity and 240 bp of complete disparity. Southern hybridization analysis suggests that both subfamilies of ETn sequences are represented many times in the mouse genome. The possibility that the disparate sequences have a role in transposition by ETn elements is discussed.     

ART-CH, a new chicken retroviruslike element.

A 3' region of a previously unknown retroviruslike element named ART-CH (avian retrotransposon from chicken genome) was obtained in the course of polymerase chain reaction-mediated cloning of avian leukosis virus long terminal repeats (LTRs) from DNAs of infected chicken cells. About 50 copies of ART-CH are present in the genome of chickens of different breeds. ART-CH is not found in DNA of quails, ducks, turkeys, or several other birds tested. The ART-CH element is about 3 kb in size, including 388 bp LTRs. The major class of ART-CH-specific RNA, also 3 kb in size, is detected in various organs of chickens. An ART-CH polypurine tract, a tRNA(Trp)-binding site, regions around the TATA box and polyadenylation signal, and the beginning of the putative gag gene strongly resemble the corresponding regions of avian leukosis viruses and EAV, the two described classes of chicken retroviruses. An open reading frame capable of encoding a polypeptide with a putative transmembrane domain is located upstream of the right ART-CH LTR. This sequence, as well as the U3 and U5 regions of the ART-CH LTR, has no obvious similarities with the corresponding parts of other known vertebrate retroviruses and retrotransposons. A short sequence upstream of the right LTR of ART-CH is very similar to sequences which flank the 3' ends of the oncogenes v-src, v-myc, v-fps, and v-crk in four different recombinant avian retroviruses and which are absent from the genomes of other studied avian retroviruses. Thus, ART-CH is a new endogenous chicken provirus that may participate in the formation of recombinant oncogenic retroviruses.     

Localization of sequences required in cis for yeast Ty1 element transposition near the long terminal repeats: analysis of mini-Ty1 elements.

In order to identify and characterize sequences within Ty1 elements which are required in cis for transposition, a series of mini-Ty1 plasmids were constructed and tested for transposition. Mini-Ty1s are deletion mutants of the Ty1-H3 element; Ty1 gene products required for transposition are supplied in trans from a helper Ty1 which has intact open reading frames but lacks a 3' long terminal repeat (LTR) and therefore cannot transpose itself. Up to 5 kilobase pairs of internal sequences of the 6-kilobase-pair-long Ty1 element can be deleted without a significant effect on transposition. The smallest mini-Ty1 element capable of transposition contains the 3' LTR and the transcribed portion of the 5' LTR, 285 base pairs (bp) of internal sequence 3' to the 5' LTR, and 23 bp of internal sequence 5' to the 3' LTR. We conclude that Ty1-encoded proteins can act in trans and that cis-acting sequences in Ty1-H3 are all within or near the LTRs. Further deletion of the 285-bp internal sequence adjacent to the 5' LTR significantly reduced transposition frequency, and the mini-Ty1 RNA produced failed to be packaged into the viruslike particles efficiently. Surprisingly, several nonhomologous cellular mRNAs were also associated with viruslike particles.     

Diversity of LTR retrotransposons and their role in genome reorganization

Current views of retrotransposons possessing long terminal repeats (LTRs) are described. The existing classification and element types isolated by genome organization are considered. Experimental data are summarized to demonstrate that the replicative cycle of a retrotransposon is not restricted to a single cell and that LTR retrotransposons are transferred between somatic cells with a rate comparable with the element transposition rate within the genome of one cell. The major mechanisms mediating the role of LTR retrotransposons in reorganization of the genome are considered with regard to the strategies of their horizontal and vertical transfer.__________Translated from Genetika, Vol. 41, No. 4, 2005, pp. 542–548.Original Russian Text Copyright © 2005 by Syomin, Ilyin.     

Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.