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PubMed2004

Characterization of two distinct RNA domains that regulate translation of the Drosophila gypsy retroelement.

The genomic RNA of the gypsy retroelement from Drosophila melanogaster exhibits features similar to other retroviral RNAs because its 5' untranslated (5' UTR) region is unusually long (846 nucleotides) and potentially highly structured. Our initial aim was to search for an internal ribosome entry site (IRES) element in the 5' UTR of the gypsy genomic RNA by using various monocistronic and bicistronic RNAs in the rabbit reticulocyte lysate (RRL) system and in cultured cells. Results reported here show that two functionally distinct and independent RNA domains control the production of gypsy encoded proteins. The first domain corresponds to the 5' UTR of the env subgenomic RNA and exhibits features of an efficient IRES (IRES(E)) both in the reticulocyte lysate and in cells. The second RNA domain that encompasses the gypsy insulator can function as an IRES in the rabbit reticulocyte lysate but strongly represses translation in cultured cells. Taken together, these results suggest that expression of the gypsy encoded proteins from the genomic and subgenomic RNAs can be regulated at the level of translation.

5' Untranslated Regions↗

PubMed2001

The long terminal repeat (LTR) sequence of marY1, a retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake, is highly conserved in various higher fungi.

marY1 is an LTR-retroelement from the homobasidiomycete Tricholoma matsutake. Nucleotide sequences that correspond to the putative U3-R region and the R-U5 region of marY1 are highly conserved in various higher fungi. Data suggest that the LTR sequence of marY1 originated early in the evolution of higher fungi and has become widely distributed. Therefore, it may be useful for the construction of an LTR-mediated transformation system in basidiomycetes.

Base Sequence↗

PubMed2004

sigma marY1, the LTR of the gypsy-type retroelement marY1 from the basidiomycete tricholoma matsutake, allows multicopy DNA integration in Lentinula edodes.

sigma marY1 is the LTR of the retroelement marY1 from the homobasidiomycete Tricholoma matsutake. Upon integration through transformation, pLC1-hph carrying a sigma marY1 derivative, sigma* marY1, conferred the hygromycin-resistant phenotype stronger than the vector without sigma* marY1 on Lentinula edodes. Based on the densitometric analysis after Southern hybridization, a copy number of the former construct integrated in the genome is much higher than that of the latter. We conclude that sigma marY1 allows multicopy DNA integration and will be useful in the genetic research on this fungal group.

Agaricales↗

PubMed2006

Significant expansion of Vicia pannonica genome size mediated by amplification of a single type of giant retroelement.

Amplification and eventual elimination of dispersed repeats, especially those of the retroelement origin, account for most of the profound size variability observed among plant genomes. In most higher plants investigated so far, differential accumulation of various families of elements contributes to these differences. Here we report the identification of giant Ty3/gypsy-like retrotransposons from the legume plant Vicia pannonica, which alone make up approximately 38% of the genome of this species. These retrotransposons have structural features of the Ogre elements previously identified in the genomes of pea and Medicago. These features include extreme size (25 kb), the presence of an extra ORF upstream of the gag-pol region, and a putative intron dividing the prot and rt coding sequences. The Ogre elements are evenly dispersed on V. pannonica chromosomes except for terminal regions containing satellite repeats, their individual copies show extraordinary sequence similarity, and at least part of them are transcriptionally active, which suggests their recent amplification. Similar elements were also detected in several other Vicia species but in most cases in significantly lower numbers. However, there was no obvious correlation of the abundance of Ogre sequences with the genome size of these species.

Base Sequence↗

PubMed2005

Cell line fingerprinting using retroelement insertion polymorphism.

Human cell lines are an indispensable tool for functional studies of living entities in their numerous manifestations starting with integral complex systems such as signal pathways and networks, regulation of gene ensembles, epigenetic factors, and finishing with pathological changes and impact of artificially introduced elements, such as various transgenes, on the behavior of the cell. Therefore, it is highly desirable to have reliable cell line identification techniques to make sure that the cell lines to be used in experiments are exactly what is expected. To this end, we developed a set of informative markers based on insertion polymorphism of human retroelements (REs). The set includes 47 pairs of PCR primers corresponding to introns of the human genes with dimorphic LINE1 (L1) and Alu insertions. Using locus-specific PCR assays, we have genotyped 10 human cell lines of various origins. For each of these cell lines, characteristic fingerprints were obtained. An estimated probability that two different cell lines possess the same marker genotype is about 10-18. Therefore, the proposed set of markers provides a reliable tool for cell line identification.

Alu Elements↗

PubMed2004

Alu and L1 retroelements are correlated with the tissue extent and peak rate of gene expression, respectively.

We exploited the serial analysis of gene expression (SAGE) libraries and human genome database in silico to correlate the breadth of expression (BOE; housekeeping versus tissue-specific genes) and peak rate of expression (PRE; high versus low expressed genes) with the density distribution of the retroelements. The BOE status is linearly associated with the density of the sense Alus along the 100 kb nucleotides region upstream of a gene, whereas the PRE status is inversely correlated with the density of antisense L1s within a gene and in the up- and downstream regions of the 0-10 kb nucleotides. The radial distance of intranuclear position, which is known to serve as the global domain for transcription regulation, is reciprocally correlated with the fractions of Alu (toward the nuclear center) and L1 (toward the nuclear edge) elements in each chromosome. We propose that the BOE and PRE statuses are related to the reciprocal distribution of Alu and L1 elements that formulate local and global expression domains.

Alu Elements↗

PubMed1997

[Structural features of the modified BARE-retroelement in the barley (Hordeum vulgare L.) genome].

The primary structure of the 4.2-kb BamHI-fragment occurring abundantly in the genome of barley Hordeum vulgare was determined. By means of computer analysis, considerable homology was found between this fragment and the copia-like BARE-l retrotransposon studied earlier. A unique distinction of the BamHI fragment is its symmetrical structure caused by the presence of two mutually inverted parts, each of which is homologous to a long region of BARE-l including a 5'-LTR (long terminal repeat) and the adjacent leader sequence. No sequences homologous to the coding domains of BARE-l were revealed in the fragment. However, potentially functional signals were found: TATA boxes and primer-binding sites (PBS) exhibiting statistically significant homology to the corresponding regulatory signals of known retroelements. Thus, we have revealed and characterized a repeated element of the H. vulgare genome that is a markedly modified derivative of the BARE family of retrotransposons of this genome.

Base Sequence↗

PubMed1994

Poly(A) site selection in the yeast Ty retroelement requires an upstream region and sequence-specific titratable factor(s) in vitro.

In the Ty retrotransposon of Saccharomyces cerevisiae, as in most retroelements, the polyadenylation site of the 5' long terminal repeat (LTR) is ignored and the one in the 3' LTR is efficiently used. We examine here the contribution to this poly(A) site selection of the region termed 'U3', corresponding to the upstream non-transcribed portion of the 5' LTR. Using an established assay in vitro, we find that 3' processing is accurate and efficient with an RNA substrate corresponding to most of the LTR, whereas none is detectable with a shorter transcript lacking the U3 region, thus explaining why the 5' poly(A) site is ignored in genomic Ty mRNA. When HIS4 coding RNA, representing 'non-specific' sequence, replaces the U3 region, the Ty polyadenylation site is activated to 50% of the wild-type level. Within one specific region (TS1) in U3, 90-95 nt upstream of the poly(A) site, the change of UAGUAU to UCGCAU reduces processing efficiency by half, to the non-specific level provided by other sequences or by a deletion of the TS1 region. Another region (TS2) near the poly(A) site appears to be independently responsible for the remaining half of the processing activity. Alteration of both TS1 and TS2 eliminates processing entirely. In competition assays, excess unlabeled U3, but not its mutated counterparts, reduces the processing of radiolabeled Ty mRNA, suggesting the involvement of some sequence-specific titratable factor(s) in the whole cell extract for U3-specific activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗

PubMed2002

Genomic and chromosomal organization of Ty1- copia-like sequences in Olea europaea and evolutionary relationships of Olea retroelements.

The Ty1- copia-like retrotransposon is one of the commonest class of transposable elements in the plant kingdom, often comprising several percent of the total DNA content. We aimed to study the evolutionary relationships of Olea retroelements, using part of the reverse transcriptase domain, as well as the genomic and chromosomal organization of these sequences in Olea europaea chromosomes and their transcription activity and copy number. Fourteen clones, that were isolated from four different species, were sequenced and a phylogenetic tree was constructed based on their predicted amino acids. Five clones derived from O. europaea were clustered together with a 87% nucleotide sequence homology and two Olea oleaster clones showed 98% sequence homology. The rest of the clones showed heterogeneity among them, leading to a common ancestral transposon that existed before the genus arose. The Ty1- copia-like sequences have a dispersed genomic organization, physically distributed on all chromosomes, showing minor clustering in some cases and low copy numbers in the smallest chromosome pair. The total copy number in the O. europaea genome was estimated by dot blotting to be 40,000 in a haploid nucleus, but a number of these are non-functional since the sequenced clones contained stop codons and frame-shifts. Some Ty1- copia-like copies, present in O. europaea, were found to be methylated, while no differences in methylation were observed between DNA isolated from young leaves and callus-suspension cultures.

Journal Article↗

PubMed2004

Highly polymorphic DNA markers to specify strains of the ectomycorrhizal basidiomycete Tricholoma matsutake based on sigmamarY1, the long terminal repeat of gypsy-type retroelement marY1.

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies--matsutake--in Pinus sp. forest. Here we report that PCR with outward facing primers designed based on sequences comprising sigma(marY1), the long terminal repeat of the gypsy-type retroelement marY1, specifies strains of T. matsutake. PCR with a primer based on the 22-bp sequence conserved at the 5'-end of sigma(marY1) conferred 73 reliable bands overall whose profiles depend upon strains of T. matsutake and T. magnivelare, the latter known as 'American matsutake'. This PCR system gave no detectable band in any other species of Tricholoma tested, including T. bakamatsutake and T. fulvocastaneum, symbionts closely related to T. matsutake, as well as a host plant, Pinus densiflora. Similarly, PCR with a set of primers based on 26-bp and 28-bp sequences at bp 48-73 and bp 281-308 of sigma(marY1), internal regions that are mutated in a variant of marY1, conferred 90 reliable bands only in strains of T. matsutake. Theoretically, PCR with the 22-bp primer would allow generation of 2(73), or 9.4x10(21), types of polymorphism, and PCR with a combination of 26- and 28-bp primers, 2(90), or 1.2x10(27) types. The probability of falsely specifying two different isolates as the same strain is <1/10(21). While polymorphisms conferred by the primer based on the 5' end of sigma(marY1) rather exhibit genetic conservation of a group of T. matsutake, those resulting from primers based on the internal sequences more clearly demonstrate intra-specific diversification. Both systems revealed that T. matsutake is divergent within the species. Ectomycorrhizas formed between P. densiflora and T. matsutake were identified by the PCR systems developed in the present study. This method, using sigma(marY1) as a genetic marker, is useful in analyzing the diversity of T. matsutake, monitoring the behavior of individual mycorrhizas, and specifying the ecological background of fruit bodies traded in markets.

Agaricales↗

PubMed1991

Retroelements in bacteria.

A peculiar type of satellite DNA, called msDNA, has been discovered in myxobacteria and some natural isolates of E. coli. These molecules are characterized by the presence of single-stranded DNA branching out from an internal guanosine residue of an RNA molecule by a unique 2',5'-phosphodiester linkage. Reverse transcriptase is required for the synthesis of msDNA. The discovery of retroelements in bacterial populations raises many intriguing questions concerning the evolutionary origin of reverse transcriptase, the function and the biosynthesis of msDNA, and the nature of the mechanisms generating the extensive diversity found in msDNA and reverse transcriptase genes among different bacterial strains.

Biological Evolution↗

PubMed1993

The retron: a bacterial retroelement required for the synthesis of msDNA.

'Retrons' are bacterial retroelements responsible for the synthesis of msDNA, a hybrid nucleic acid consisting of a single-stranded DNA that is branched out from an internal guanosine of an RNA molecule via a 2',5'-phosphodiester linkage. Retrons are found in a minor population of various bacterial species and are extensively diverse. Two important questions now demanding attention are whether retrons are mobile elements and why are they so diverse?

Base Sequence↗

PubMed1995

Phylogenetic analyses of 55 retroelements on the basis of the nucleotide and product amino acid sequences of the pol gene.

Comparisons of pol gene nucleotide and reverse transcriptase (RT) amino acid sequences of 47 retroviruses, 3 caulimoviruses, and 5 hepadnaviruses showed that approximately one-third of the gene at the 5' end is much more conserved than other pol regions. The most conserved regions on both the nucleotide and amino acid sequences were chosen for construction of phylogenetic trees. The maximum-parsimony and distance-matrix methods were used for analyses of aligned amino acid sequences; these two methods, and the compatibility method, were used to analyze the aligned nucleotide sequences. Essentially identical majority-rule consensus trees were produced by these different methods from both the pol gene nucleotide and RT amino acid sequences, which divided the 55 retroelements into six major groups. The reliability of the phylogenetic trees was probed with the bootstrapping of 100 replicates of the original sequence alignments. The grouping results were shown to be statistically significant by multiple comparisons with the least-significant-difference procedure.

Amino Acid Sequence↗

PubMed1991

Association of a retroelement with a P4-like cryptic prophage (retronphage phi R73) integrated into the selenocystyl tRNA gene of Escherichia coli.

A new multicopy single-stranded DNA (msDNA-Ec73) was found in a clinical strain of Escherichia coli. Retron-Ec73, consisting of an msDNA-coding region and the gene for reverse transcriptase (RT), was found to be a part of a 12.7-kb foreign DNA fragment flanked by 29-bp direct repeats and integrated into the gene for selenocystyl-tRNA (selC) at 82 min on the E. coli chromosome. Except for the 2.4-kb retron region, the integrated DNA fragment showed remarkable homology to most of the bacteriophage P4 genome. Among the phage genes found in this element, however, the integrase gene had very low identity (40%) to P4 integrase, indicating that the cryptic prophage associated with the retroelement has its own unique site-specific integrase different from P4 integrase. Recently, we have shown that P2 phage can act as a helper to excise the cryptic prophage and to package its genome into an infectious virion. The newly formed phage (retronphage phi R73) can also lysogenize a new host strain, reintegrating its genome into the selC gene and enabling the newly formed lysogen to produce msDNA-Ec73 (S. Inouye, M. G. Sunshine, E. W. Six, and M. Inouye, Science 252:969-971, 1991).

Amino Acid Sequence↗

PubMed1994

Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements.

Integrons confer on bacterial plasmids a capability of taking up antibiotic resistance genes by integrase-mediated recombination. We show here that integrons are situated on genetic elements flanked by 25-bp inverted repeats. The element carrying the integron of R751 has three segments conserved with similar elements in Tn21 and Tn5086. Several characteristics suggest that this element is a transposon, which we call Tn5090. Tn5090 was shown to contain an operon with three open reading frames, of which two, tniA and tniB, were predicted by amino acid similarity to code for transposition proteins. The product of tniA (559 amino acids) is a probable transposase with 25% amino acid sequence identity to TnsB from Tn7. Both of these polypeptides contain the D,D(35)E motif characteristic of a protein family made up of the retroviral and retrotransposon IN proteins and some bacterial transposases, such as those of Tn552 and of a range of insertion sequences. Like the transposase genes in Tn552, Mu, and Tn7, the tniA gene was followed by a gene, tniB, for a probable ATP-binding protein. The ends of Tn5090, like those of most other elements producing D,D(35)E proteins, begin by 5'-TG and also contains a complex structure with four 19-bp repeats at the left end and three at the right end. Similarly organized repeats have been observed earlier at the termini of both Tn7 and phage Mu, where they bind their respective transposases and have a role in holoenzyme assembly. Another open reading frame observed in Tn5090, tniC, codes for a recombinase of the invertase/resolvase family, suggesting a replicative transposition mechanism. The data presented here suggest that Tn5090, Tn7, Tn552, and Mu form a subfamily of bacterial transposons which in parallel to many insertion sequences are related to the retroelements.

Amino Acid Sequence↗

PubMed1995

Full-length proviruses of baboon endogenous virus (BaEV) and dispersed BaEV reverse transcriptase retroelements in the genome of baboon species.

Baboon endogenous virus (BaEV) is present in multiple copies in many Old World monkey species. BaEV proviruses may contain open reading frames for all major genes, as is indicated by the rescue of infectious virus particles from baboon and gelada tissues after cocultivation with permissive cells. We have analyzed full-length BaEV proviral structures in a baboon (Papio cynocephalus) genomic library and found no evidence for the rearrangements or large deletions commonly observed in endogenous virus genomes from other mammalian species. The two proviruses studied were integrated next to or nearby long interspersed repeat sequence (LINE) transposable elements. Additionally, isolated dispersed fragments with 100% and approximately 77% homology, respectively, to part of the BaEV reverse transcriptase gene were detected. These presumed retroelements were present in an approximately 10-fold excess compared with the full-length proviral genomes. PCR amplification and sequencing of BaEV reverse transcriptase and env fragments from the lambda clones and from the genomic DNA of other baboon species showed that there is little sequence variation present in BaEV DNA in the baboon genome.

Animals↗

PubMed2001

marY2N, a LINE-like non-long terminal repeat (non-LTR) retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake.

A LINE-like non-LTR retroelement designated marY2N was cloned from the ectomycorrhizal homobasidiomycete Tricholoma matsutake. marY2N has open reading frames that correspond to gag and pol, and a putative promoter and consensus sequences common to those of the mutators from fruit flies. While it is common to T. matsutake and Tricholoma magnivelare, marY2N does not reside in any other species of Tricholoma tested.

Base Sequence↗

PubMed2001

Expression of marY1, a gypsy-type LTR-retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake, in the budding yeast Saccharomyces cerevisiae.

marY1 is a gypsy-type LTR-retroelement present in the genome of the ectomycorrhizal homobasidiomycete Tricholoma matsutake. We document here that a marY1-lacZ gene fusion was expressed in the budding yeast Saccharomyces cerevisiae. The finding strongly suggests that marY1 is activated by trans-regulatory factors common to higher fungi, and may be useful for the development of new recombinant systems in ectomycorrhizal fungi and homobasidiomycetes.

Base Sequence↗