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At least 577 records · Page 32Linked to original sources
PubMed2006

Analysis of retrotransposon structural diversity uncovers properties and propensities in angiosperm genome evolution.

Analysis of LTR retrotransposon structures in five diploid angiosperm genomes uncovered very different relative levels of different types of genomic diversity. All species exhibited recent LTR retrotransposon mobility and also high rates of DNA removal by unequal homologous recombination and illegitimate recombination. The larger plant genomes contained many LTR retrotransposon families with >10,000 copies per haploid genome, whereas the smaller genomes contained few or no LTR retrotransposon families with >1,000 copies, suggesting that this differential potential for retroelement amplification is a primary factor in angiosperm genome size variation. The average ratios of transition to transversion mutations (Ts/Tv) in diverging LTRs were >1.5 for each species studied, suggesting that these elements are mostly 5-methylated at cytosines in an epigenetically silenced state. However, the diploid wheat Triticum monococcum and barley have unusually low Ts/Tv values (respectively, 1.9 and 1.6) compared with maize (3.9), medicago (3.6), and lotus (2.5), suggesting that this silencing is less complete in the two Triticeae. Such characteristics as the ratios of point mutations to indels (insertions and deletions) and the relative efficiencies of DNA removal by unequal homologous recombination compared with illegitimate recombination were highly variable between species. These latter variations did not correlate with genome size or phylogenetic relatedness, indicating that they frequently change during the evolutionary descent of plant lineages. In sum, the results indicate that the different sizes, contents, and structures of angiosperm genomes are outcomes of the same suite of mechanistic processes, but acting with different relative efficiencies in different plant lineages.

Animals↗
PubMed1999

Isolation and characterization of ARA160 as the first androgen receptor N-terminal-associated coactivator in human prostate cells.

The androgen receptor (AR) is a member of the steroid receptor superfamily that may require coactivators for proper or maximal transactivation. Using a purified AR N-terminal peptide as a probe to screen the human testis expression library, we identified an androgen-enhanced AR N-terminal-associated protein ARA160, which consists of 1,093 amino acids with an apparent molecular mass of 160 kDa. Sequence comparison in GenBank(TM) reveals that ARA160 shares an identical sequence with a HIV-1 TATA element modulatory factor, TMF. The far-Western blotting and co-immunoprecipitation assays demonstrate that the AR can interact directly with ARA160/TMF. Affinity gel pull-down and mammalian two-hybrid assays further suggest androgen can enhance significantly the interaction between AR and ARA160. Transient transfection assays demonstrated that ARA160 might function as a coactivator for AR-mediated transactivation in human prostate cancer PC-3 cells. Our data further suggest that this AR N-terminal coactivator can function cooperatively with AR C-terminal coactivator, ARA70, in PC-3 cells. Together, our data demonstrate that ARA160 might represent the first identified androgen-enhanced N-terminal coactivator for the AR.

Amino Acid Sequence↗
PubMed2005

Phylogenetic heterogeneity of new HTLV type 1 isolates from southern India in subgroup A.

Seven isolates of human T cell leukemia virus type 1 (HTLV-1) were taken in southern India and phylogenetically analyzed to gain new insights into the origin and dissemination of HTLV-1 in the subcontinent. The new Indian HTLV-1s were found to be members of subgroup A (Transcontinental subgroup) of the Cosmopolitan group. They formed three different clusters (South African/Caribbean, Middle Eastern, and East Asian clusters). These results demonstrate that Indian HTLV-1s are genetically heterogeneous and include the most divergent strain of subgroup A. On the basis of these results, we speculate that subgroup A HTLV- 1s may have been present for thousands of years in India.

Adult↗
PubMed2003

GABI-Kat SimpleSearch: a flanking sequence tag (FST) database for the identification of T-DNA insertion mutants in Arabidopsis thaliana.

SUMMARY: GABI-Kat SimpleSearch is a database of flanking sequence tags (FSTs) of T-DNA mutagenized Arabidopsis thaliana lines that were generated by the GABI-Kat project. Sequences flanking the T-DNA insertion sites were aligned to the A.thaliana genome sequence, annotated with information about the FST, the insertion site and the line from which the FST was derived. A web interface permits text-based as well as sequence-based searches for relevant insertions. GABI-Kat SimpleSearch aims to help biologists to quickly find T-DNA insertion mutants for their research. AVAILABILITY: http://www.mpiz-koeln.mpg.de/GABI-Kat/

Arabidopsis↗
PubMed2004

Retrotransposon-gene associations are widespread among D. melanogaster populations.

We have surveyed 18 natural populations of Drosophila melanogaster for the presence of 23 retrotransposon-gene-association alleles (i.e., the presence of an LTR retrotransposon sequence in or within 1,000 bp of a gene) recently identified in the sequenced D. melanogaster genome. The identified associations were detected only in the D. melanogaster populations. The majority (61%) of the identified retrotransposon-gene associations were present only in the sequenced strain in which they were first identified. Thirty percent of the associations were detected in at least one of the natural populations, and 9% of the associations were detected in all of the D. melanogaster populations surveyed. Sequence analysis of an association allele present in all populations indicates that selection is a significant factor in the spread and/or maintenance of at least some of retroelement-gene associations in D. melanogaster.

Alleles↗
PubMed2004

Germline transformation of the butterfly Bicyclus anynana.

Ecological and evolutionary theory has frequently been inspired by the diversity of colour patterns on the wings of butterflies. More recently, these varied patterns have also become model systems for studying the evolution of developmental mechanisms. A technique that will facilitate our understanding of butterfly colour-pattern development is germline transformation. Germline transformation permits functional tests of candidate gene products and of cis-regulatory regions, and provides a means of generating new colour-pattern mutants by insertional mutagenesis. We report the successful transformation of the African satyrid butterfly Bicyclus anynana with two different transposable element vectors, Hermes and piggyBac, each carrying EGFP coding sequences driven by the 3XP3 synthetic enhancer that drives gene expression in the eyes. Candidate lines identified by screening for EGFP in adult eyes were later confirmed by PCR amplification of a fragment of the EGFP coding sequence from genomic DNA. Flanking DNA surrounding the insertions was amplified by inverse PCR and sequenced. Transformation rates were 5% for piggyBac and 10.2% for Hermes. Ultimately, the new data generated by these techniques may permit an integrated understanding of the developmental genetics of colour-pattern formation and of the ecological and evolutionary processes in which these patterns play a role.

Animals↗
PubMed2001

Highly conserved sequences flank avirulence genes: isolation of novel avirulence genes from Pseudomonas syringae pv. pisi.

DNA sequences flanking two avr genes (avrPpiA1 and avrPpiB1) from Pseudomonas syringae pv. pisi show a high degree of similarity. Specific primers designed from the conserved regions were used in PCR amplifications with all P. syringae pv. pisi races. As well as amplifying the expected avrPpiA- and avrPpiB-containing fragments, two additional fragments were amplified: one contained a single open reading frame (ORF1) and was found in races of genomic group II (2, 3A, 4A and 6); the second fragment contained two open reading frames (ORF2 and ORF3), separated by 658 nt, and was detected in all races. All three ORFs had G+C ratios (46.9-48 mol%) that were significantly less than that for P. syringae and each was preceded by a potential hrp box promoter. In P. syringae pv. phaseolicola, ORF1 and ORF2 each elicited a strong non-host hypersensitive reaction on bean leaves; ORF1 was designated avrPpiG, the product of which had strong similarity to AvrRxv, AvrBsT and YopP. ORF2 was identical to a gene, designated avrPpiC, previously isolated from P. syringae pv. pisi race 5. ORF3 was always found in association with avrPpiC and both were detected in a wide range of P. syringae pathovars. In contrast, avrPpiG was only detected in strains of P. syringae pv. pisi genomic group II and P. syringae pv. coronafaciens (ICMP 3113). In P. syringae pv. pisi, avrPpiG was plasmid-borne and avrPpiC and ORF3 were chromosomal. This conservation of flanking sequences has implications for the horizontal transfer of avirulence and virulence genes, suggesting that specific regions of the bacterial genome act as sites for their integration/excision.

Bacterial Proteins↗
PubMed2004

Characterization of germline porcine endogenous retroviruses from Large White pig.

Porcine endogenous retroviruses (PERV) are of concern when the microbiological safety aspects of xenotransplantation are considered. Four unique isolates of PERV B have been identified previously from a lambda library constructed from genomic DNA from a Large White pig. This study shows that none of these isolates are replication competent when transfected into permissive human or pig cells in vitro, and the removal of flanking genomic sequences does not confer a human tropic replication competent (HTRC) phenotype on these PERV proviruses. Analysis of the envelope sequences revealed that PERV B demonstrated high similarity to the envelope sequences derived from replication-competent PERV, indicating that lack of replication competence does not appear to be attributable to this region of the provirus. These data complement recent findings that HTRC PERV are recombinants between the PERV A and PERV C subgroups, and that these recombinants are not present in the germline of miniature swine. Together, these results indicate that these individual PERV B proviruses are unlikely to give rise to HTRC PERV.

Amino Acid Sequence↗
PubMed2002

Construction of a self-excisable bacterial artificial chromosome containing the human cytomegalovirus genome and mutagenesis of the diploid TRL/IRL13 gene.

The full-length genome of human cytomegalovirus strain AD169 was cloned as an infectious bacterial artificial chromosome (BAC) plasmid, pAD/Cre. The BAC vector, flanked by LoxP sites, was inserted immediately after the Us28 open reading frame without deletion of any viral sequences. The BAC vector contained the Cre recombinase-encoding gene disrupted by an intron under control of the simian virus 40 early promoter. When pAD/Cre was transfected into primary human foreskin fibroblast cells, Cre was expressed and mediated site-specific recombination between the two LoxP sites, excising the BAC DNA backbone. This gave rise to progeny virus that was wild type with the exception of an inserted 34-bp LoxP site. We performed site-directed mutagenesis on pAD/Cre to generate a series of viruses in which the TRL/IRL13 diploid genes were disrupted and subsequently repaired. The mutants reach the same titer as the wild-type virus, indicating that the TRL/IRL13 open reading frames are not required for virus growth in cell culture. The sequence of the TRL13 open reading frame in the low-passage Toledo strain of human cytomegalovirus is quite different from the corresponding region in the AD169 strain. One of multiple changes is a frameshift mutation. As a consequence, strain Toledo encodes a putative TRL13 protein whose C-terminal domain is larger (extending through the TRL14 coding region) and encodes in a reading frame different from that of strain AD169. We speculate that the strain AD169 coding region has drifted during passage in the laboratory. We propose that TRL13 has been truncated in strain AD169 and that the partially overlapping TRL14 open reading frame is not functional. This view is consistent with the presence of both TRL13 and -14 on all mRNAs that we have mapped from this region, an organization that would include the much longer strain Toledo TRL13 open reading frame on the mRNAs.

Amino Acid Sequence↗
PubMed2003

High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines.

A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline included robotized extraction of genomic DNA in a 96-well format, an adapter-ligation PCR method for amplification of plant sequences adjacent to T-DNA borders, automated purification and sequencing of PCR products, and computational trimming of the resulting sequence files. Data quality was significantly improved by (i) restriction digestion of the adaptor-ligation products to reduce trivial sequences caused by co-amplification of fragments derived from the free plasmid, and (ii) the design of the adaptor primers for the second amplification step to enhance selective generation of single PCR fragments, even from lines with multiple T-DNA insertions. Gel-purification was avoided by including these steps, the number of amplification reactions per line was reduced from four to three, and the percentage of lines that yielded at least one FST was increased from 66% to 86%. More than 58,000 FSTs have been submitted to GenBank and are available at http://www.mpiz-koeln.mpg.de/GABI-Kat/.

Arabidopsis↗
PubMed1995

Tat mediates transcriptional activation of HIV-1 gene in vitro.

Gene expression of human immunodeficiency virus (HIV-1) is greatly enhanced by a viral transactivator, the Tat protein, which interacts with R region sequences of the HIV-1 long terminal repeat (LTR). There is no direct evidence to indicate transcriptional activation of HIV-1 by Tat. Using an in vitro transcription system, we demonstrate that an established mouse cell line, which constitutively expresses Tat protein, selectively stimulates the steady state levels of the transcripts directed from the long terminal repeat (LTR) sequences of HIV-1. The gel binding retardation assays further demonstrate a stable activated complex, formed due to direct binding of Tat to DNA elements of the HIV-1 LTR. These data implicate transcription as the site of Tat action in trans-activation and could play an essential role in human immunodeficiency virus replication, similar to the nuclear trans-activators of other viruses.

Animals↗
PubMed1999

IS1630 of Mycoplasma fermentans, a novel IS30-type insertion element that targets and duplicates inverted repeats of variable length and sequence during insertion.

A new insertion sequence (IS) of Mycoplasma fermentans is described. This element, designated IS1630, is 1,377 bp long and has 27-bp inverted repeats at the termini. A single open reading frame (ORF), predicted to encode a basic protein of either 366 or 387 amino acids (depending on the start codon utilized), occupies most of this compact element. The predicted translation product of this ORF has homology to transposases of the IS30 family of IS elements and is most closely related (27% identical amino acid residues) to the product of the prototype of the group, IS30. Multiple copies of IS1630 are present in the genomes of at least two M. fermentans strains. Characterization and comparison of nine copies of the element revealed that IS1630 exhibits unusual target site specificity and, upon insertion, duplicates target sequences in a manner unlike that of any other IS element. IS1630 was shown to have the striking ability to target and duplicate inverted repeats of variable length and sequence during transposition. IS30-type elements typically generate 2- or 3-bp target site duplications, whereas those created by IS1630 vary between 19 and 26 bp. With the exception of two recently reported IS4-type elements which have the ability to generate variable large duplications (B. B. Plikaytis, J. T. Crawford, and T. M. Shinnick, J. Bacteriol. 180:1037-1043, 1998; E. M. Vilei, J. Nicolet, and J. Frey, J. Bacteriol. 181:1319-1323, 1999), such large direct repeats had not been observed for other IS elements. Interestingly, the IS1630-generated duplications are all symmetrical inverted repeat sequences that are apparently derived from rho-independent transcription terminators of neighboring genes. Although the consensus target site for IS30 is almost palindromic, individual target sites possess considerably less inverted symmetry. In contrast, IS1630 appears to exhibit an increased stringency for inverted repeat recognition, since the majority of target sites had no mismatches in the inverted repeat sequences. In the course of this study, an additional copy of the previously identified insertion sequence ISMi1 was cloned. Analysis of the sequence of this element revealed that the transposase encoded by this element is more than 200 amino acid residues longer and is more closely related to the products of other IS3 family members than had previously been recognized. A potential site for programmed translational frameshifting in ISMi1 was also identified.

Amino Acid Sequence↗
PubMed1987

Specific binding of transposase to terminal inverted repeats of transposable element Tn3.

Tn3 transposase, which is required for transposition of Tn3, has been purified by a low-ionic-strength-precipitation method. Using a nitrocellulose filter binding assay, we have shown that transposase binds to any restriction fragment. However, binding of the transposase to specific fragments containing the terminal inverted repeat sequences of Tn3 can be demonstrated by treatment of transposase-DNA complexes with heparin, which effectively removes the transposase bound to the other nonspecific fragments at pH 5-6. DNase I "footprinting" analysis showed that the transposase protects an inner 25-base-pair region of the 38-base-pair terminal inverted repeat sequence of Tn3. This protection is not dependent on pH. Interestingly, binding of the transposase to the inverted repeat sequences facilitates DNase I to nick at the end of the Tn3 sequence. It was also observed that the transposase protects the end regions of restriction fragments with a cohesive sequence at their 5' end or with a flush end from DNase I cleavage. The specific and nonspecific binding of transposase to DNA is ATP-independent.

Adenosine Triphosphate↗
PubMed1986

Expression and characterization of the trans-activator of HTLV-III/LAV virus.

The human T-lymphotropic retrovirus HTLV-III/LAV encodes a trans-activator that increases viral gene expression. We expressed this trans-activator in animal cells and studied its structural and functional characteristics. The putative trans-activator protein was immunoprecipitated from overproducing stable cell lines and shown to migrate as a 14-kilodalton polypeptide on sodium dodecyl sulfate-polyacrylamide gels. S1 nuclease mapping experiments showed that the trans-activator increases the levels of steady-state messenger RNA transcribed from the viral long terminal repeat promoter. Sequences within the R region of the HTLV-III/LAV long terminal repeat are essential for trans-activation. Quantitations of messenger RNA and protein showed that the protein increase was greater than the messenger RNA increase in CV1 and HeLa cells, indicating that more than one mechanism was responsible for the trans-activation and that cell type-specific factors may determine the final level of trans-activation.

Cell Line↗
PubMed1996

[Effect of specific nucleotide sequences on the transcription activity of long terminal repeats of avian retroviruses].

Influence of specific nucleotide sequences on the constitutive transcriptional activity of the Rous sarcoma long terminal repeats (LTR) was studied. The possible function of different elements of the transcriptional control region of the avian retroviral LTR was discussed on the basis of a comparative analysis of the effect of specific nucleotide insertions on LTR activity in vivo and in vitro.

Avian Sarcoma Viruses↗
PubMed2001

Antisense RNA regulation of the par post-segregational killing system: structural analysis and mechanism of binding of the antisense RNA, RNAII and its target, RNAI.

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA regulated post-segregational killing system (PSK) identified in a Gram-positive organism. Par encodes two small, convergently transcribed RNAs, designated RNAI and RNAII, which are the toxin and antitoxin of the par PSK system respectively. RNAI encodes an open reading frame for a 33 amino acid toxin called Fst. Expression of fst is regulated post-transcriptionally by RNAII. RNAII interacts with RNAI by a unique antisense RNA mechanism involving binding at the 5' and 3' ends of both RNAs. Par RNA interaction requires a complementary transcriptional terminator stem-loop and a set of direct repeat sequences, DRa and DRb, located at the 5' end of both RNAs. The secondary structures of RNAI, RNAII and the RNAI-RNAII complex were analysed by partial digestion with Pb(II) and ribonucleases. Probing data for RNAI and RNAII are consistent with previously reported computer generated models, and also confirm that complementary direct repeat and terminator sequences are involved in the formation of the RNAI-RNAII complex. Mutant par RNAs were used to show that the binding reaction occurs in at least two steps. The first step is the formation of an initial kissing interaction between the transcriptional terminator stem-loops of both RNAs. The subsequent step(s) involves an initial pairing of the complementary direct repeat sequences followed by complete hybridization of the 5' nucleotides to stabilize the RNAI-RNAII complex.

Amino Acid Sequence↗