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At least 775 records · Page 43Linked to original sources
PubMed2002

Integration site selection by lentiviruses: biology and possible control.

Retroviruses integrate into naked DNA in a generally sequence nonspecific fashion, but closer study reveals a variety of forces that influence target site selection. Primary sequence of the target plays a small but detectable role. Proteins bound to target DNA can inhibit integration by blocking access of integration complexes or stimulate integration by distorting DNA. An important example of the latter is DNA distortion in nucleosomal DNA. In vivo integration has not yet been convincingly shown to be biased in favor of any identifiable sequence features, though this could still change in future studies. Many applications of retroviral vectors could be facilitated by targeting integration in vivo to predetermined sites. Towards this end, several groups have studied the properties of fusions of integrase proteins to sequence-specific DNA-binding domains. To date such studies establish that targeting can work well in reactions in vitro, but a variety of obstacles complicate applications in vivo. However, naturally occurring retrotransposons do carry out highly targeted integration using retrovirus-like integrase proteins, fueling long-term hopes for targeting with retroviral integrases as well.

DNA, Viral↗
PubMed1995

The maize GapC4 promoter confers anaerobic reporter gene expression and shows homology to the maize anthocyanin regulatory locus C1.

The cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC) gene family of maize is differentially expressed in response to anaerobic stress. While GapCl and GapC2 are downregulated, GapC3 and GapC4 are anaerobically induced. We have sequenced and analyzed a 3073 bp promoter fragment of GapC4. The promoter confers anaerobic induction of a reporter gene construct in a transient gene expression system in maize. Deletion analysis of the GapC4 promoter revealed a 270 bp long DNA region required for anaerobic induction. This region contains sequence motifs resembling the cis-acting sequences of the anaerobically induced maize Adh1 and Adh2 genes. Furthermore, the 3073 bp GapC4 promoter fragment displays homology to long terminal repeats of maize retrotransposons and to the 3' region of the maize anthocyanin regulatory locus C1.

Anaerobiosis↗
PubMed1996

A transcriptional analysis of the S1Bn (Brassica napus) family of SINE retroposons.

S1Bn is a plant short interspersed element (SINE) whose amplification probably involves the reverse transcription of an RNA intermediate. In this report, we identified and characterized S1Bn transcripts from different Brassica napus tissues. Despite the presence of a consensus internal POL III promoter in a large number of genomic S1Bn elements, we observed that S1Bn transcripts are rare in B. napus cells. The use of two very sensitive methods (RT-PCR and RACE PCR) allowed the characterization of 102 independent S1Bn cDNA clones from three different tissues (shoot, root and callus). From this analysis, we conclude that the majority of S1Bn transcripts probably result from a small number of cotranscriptional events where an S1Bn element is transcribed due to its presence in a POL II transcriptional unit. Specific POL III RNA transcripts, initiating at the first 5' nucleotide of the DNA element, are also present in the tested tissues and possibly result from the transcriptional activity of as few as three genomic elements. Two of these transcripts could represent master transcripts responsible for the amplification of S1Bn subfamilies. We also observed that the population of specific POL III transcripts varies among the three tested tissues and that some transcripts appear completely tissue-specific.

Base Sequence↗
PubMed1996

The BARE-1 retrotransposon is transcribed in barley from an LTR promoter active in transient assays.

The BARE-1 retrotransposon occurs in more than 10(4) copies in the barley genome. The element is bounded by long terminal repeats (LTRs, 1829 bp) containing motifs typical of retrotransposon promoters. These, the presence of predicted priming sites for reverse transcription, and the high conservation for all key functional domains of the coding region suggest that copies within the genome could be active retrotransposons. In view of this, we looked for transcription of BARE-1 within barley tissues and examined the promoter function of the BARE-1 LTR. We demonstrate here that BARE-1-like elements are transcribed in barley tissues, and that the transcripts begin within the BARE-1 LTR downstream of TATA boxes. The LTR can drive expression of reporter genes in transiently transformed barley protoplasts. This is dependent on the presence of a TATA box functional in planta as well. Furthermore, we identify regions within the LTR responsible for expression within protoplasts by deletion analyses of LTR-luc constructs. Similarities between promoter regulatory motifs and regions of the LTR were identified by comparisons to sequence libraries. The activity of the LTR as a promoter, combined with the abundance of BARE-1 in the genome, suggests that BARE-1 may retain the potential for propagation in the barley genome.

Base Sequence↗
PubMed1994

Expression of the tobacco Tnt1 retrotransposon promoter in heterologous species.

The expression of the tobacco (Nicotiana tabacum) retrotransposon Tnt1 has previously been shown to be strongly regulated and driven from the 5' long terminal repeat (LTR). We report here that the Tnt1 LTR can promote activity of the beta-glucuronidase (GUS) reporter gene in two heterologous species of the Brassicaceae family, namely rapeseed (Brassica napus) and Arabidopsis thaliana. The translational LTR-GUS fusion was active in transient expression studies performed with tobacco and rapeseed protoplasts, indicating that the LTR sequences are recognized in heterologous species. Our results also showed that Tnt1 LTR-promoted GUS expression in transgenic Arabidopsis is strongly regulated, and that, in contrast to tobacco, hormonal activation plays a significant role in the expression of the Tnt1 LTR in Arabidopsis. LTR sequences were shown to be more effective than the CaMV 35S enhancer region in transient expression studies performed with tobacco or rapeseed protoplasts, and substitution of the LTR sequences upstream from the major transcriptional start with the CaMV 35S enhancer region gave high levels of expression in transgenic tobacco and Arabidopsis leaves, suggesting that a Tnt1 element with similar substitutions in its 5' LTR might be suited for gene-tagging experiments in heterologous species.

Arabidopsis↗
PubMedDate not supplied

Evidence for a regulatory protein complex on RNA polymerase III promoter of human retroposons of Alu family.

Abundant human retroposons of the Alu family produce few RNA polymerase III (RPIII)-dependent transcripts in vivo. This suggests that either the bulk of the repeats has no proper promoter elements or that transcription of Alu by RPIII is repressed. In this study, we analyzed complexes formed by human nuclear proteins with the Alu B-box and with an adjacent downstream sequence (DB-sequence). Four complexes (C1-C4) were detected and two of them (C2 and C3) were found to be induced by different proteins. C3 formation was found to be sensitive to minor sequence variation within the Alu DB-sequence. The C2 complex is specifically repressed by the competing VA1 B-box oligonucleotide and was found to be very stable. In addition, it is downregulated in human cells transformed by adenovirus 5. This is consistent with a view that the C2 complex is formed by a protein (designated as ACR1) that is different from TFIIIC2. The ACR1 protein may be involved in the modulation of Alu transcription in vivo by interfering or cooperating with TFIIIC2. A similar complex is detected with the efficiently transcribed adenovirus VA1 RNA gene B-box. We compared the affinity of complexes formed by ACR1 with Alu and VA1 B-boxes. It was found that both B-boxes bind ACR1 with equal affinity with a dissociation constant of about 2 nM. However, DB-sequences in Alu and VA1 promoters are non-homologous, and C3/C4 complexes are found to be formed with Alu DB, but not formed with VA1 DB sequences. The Alu-specific protein forming C3 (named as ACR2) may cooperate with ACR1 in selective repression of RPIII-dependent Alu transcription in vivo.

Cell Line↗
PubMed1996

Increased concentration of some transcription factor binding sites in human retroposons of the Alu family.

Eukaryotic gene expression is dependent on short protein-binding DNA sequence motifs promoting the assembly of multiprotein transcription complexes. Human retroposons of the Alu family are known to contain some high-affinity binding sites for transcription factors, which may serve as signals in regulation of expression of RNA-polymerase II-transcribed genes. In this computer study we have compared the density of ten consensus transcription factor binding sites in a set of human mature mRNA, human promotors and Alu repeats. Our results indicate that Alu retroposons and promotor sequences have significantly higher mean density of these sites compared to RNAs. It is suggested that the majority of Alu repeats do have the potential for regulating gene expression via modulation of RNA polymerase II-dependent transcription.

Base Composition↗
PubMed1996

412-positive mesodermal cells and the gonadal mesoderm are separate from the fat-cell lineage.

The Drosophila retrotransposon, 412, is expressed in a cell-specific manner during embryogenesis. At stage 11, 412 transcripts are present in bilateral clusters of cells within the mesoderm. The posterior clusters of 412-positive cells become associated with the gonads at stage 13; however, the fate of the cells in the remaining clusters is unknown. We have tested by in situ hybridization to whole-mount embryos the possible identity of these cells with known precursor cell types present in bilateral clusters. We simultaneously located the 412-positive cells and the precursor cells to visceral muscle or the fat body. We have determined that the 412-positive cells do not correspond to these precursor cells and that the development of the visceral muscle or fat body does not affect the expression of 412 during embryogenesis.

Adipocytes↗
PubMed1994

An analysis of retroposition in plants based on a family of SINEs from Brassica napus.

The identification of a family of SINE retroposons dispersed in the genome of oilseed rape Brassica napus has provided the basis for an evolutionary analysis of retroposition in plants. The repetitive elements (called S1Bn) are 170 bp long and occupy roughly 500 loci by haploid genome. They present characteristic features of SINE retroposons such as a 3' terminal A-rich region, two conserved polymerase III motifs (box A and B), flanking direct repeats of variable sizes, and a primary and secondary sequence homology to several tRNA species. A consensus sequence was made from the alignment of 34 members of the family. The retroposon population was divided into five subfamilies based on several correlated sets of mutations from the consensus. These precise separations in subfamilies based on "diagnostic" mutations and the random distribution of mutations observed inside each subfamily are consistent with the master sequence model proposed for the dispersion of mammalian retroposons. An independent analysis of each subfamily provides strong evidence for the coexpression of at least three subfamily master sequences (SMS). In contrast to mammalian retroposition, diagnostic positions are not shared between SMS. We therefore propose that SMS were all derived from a general master sequence (GMS) and independently activated for retroposition after a variable period of random drift. Possible models for plant retroposition are discussed.

Base Sequence↗
PubMed1996

Retrotransposon Mys was active during evolution of the Peromyscus leucopus-maniculatus complex.

Mys is a retrovirus-like transposable element found throughout the genus Peromyscus. Several mys subfamilies identified on the basis of restriction site variation occur in more than one species. The distribution of these subfamilies is consistent with the accepted species phylogeny, suggesting that mys was present in the ancestor of Peromyscus and has been active through much of the evolution of this genus. Quantitative Southern blot analysis was used to examine the variability of subfamilies in P. leucopus and maniculatus. We found that subfamilies with phylogenetically narrow distributions were more variable in copy number both within and between species than subfamilies with a broader distribution. Taken together, our data suggest that mys has undergone multiple rounds of transposition since the peromyscine radiation, and that five subfamilies have been amplified during the evolution of the leucopus-maniculatus species complex.

Animals↗
PubMed1994

The microinjected Drosophila melanogaster 1731 retrotransposon is activated after the midblastula stage of the amphibian Pleurodeles waltl development.

The entire 1731 retrotransposon of Drosophila melanogaster, tagged with the E. coli lac Z gene inserted in its gag sequence, was injected into oocytes and fertilized eggs of the urodele amphibian Pleurodeles waltl. Expression of the reporter gene indicated that the 1731 promoter (its 5'LTR) is active in the embryos and not in the oocytes. It appeared that this element is regulated as amphibian genes are at the beginning of the development, i.e. that expression was detected after the mid blastula stage and maintained up to four or five days after injection. Another construction associating the modified 1731 promoter with the CAT gene is also expressed in Pleurodeles embryos during the same period of development. This indicated that the 1731 promoter issued from a Drosophila species is activated as promoting sequences of amphibian zygotic genes are, suggesting that in the case of horizontal transfer, 1731 can be expressed into vertebrate organisms.

Animals↗
PubMed1995

Gypsy/Ty3-class retrotransposons integrated in the DNA of herring, tunicate, and echinoderms.

Eight new examples of retrotransposons of the Gypsy/Ty3 class have been identified in marine species. A 525-nt pol gene-coding region was amplified using degenerate primers from highly conserved regions and has extended the range of recognition of Gypsy/Ty3 far beyond those previously known. The following matrix shows the percentage AA divergence of the translations of this segment of the pol gene coding region. [table: see text] The underlines separate three groups of retrotransposons that can be recognized on the basis of this amino acid sequence. The new upper group shows surprising amino acid sequence similarity among members from the DNA of herring, sea urchin, starfish, and a tunicate. For example, the herring element differs by only 41% from the Ciona element and 46% from the sea urchin element. The group between the lines includes members close to previously known elements (marked by asterisks) and has so far been found only in sea urchins. The two upper groups differ from each other by 55-60% and yet members of both groups (e.g., Spr1 and Spr2) are integrated into the DNA of one species--S. purpuratus. Below the lower underline is listed the only known representative of a very distant group, which occurs in starfish DNA. In spite of large divergence, amino acid sequence comparisons indicate that all of the elements shown in the array are members of the LTR-containing class of retrotransposons that includes Gypsy of Drosophila and Ty3 of yeast. Of all known mobile elements this class shows the closest sequence similarity to retroviruses and has the same arrangement of genes as simpler retroviruses.

Amino Acid Sequence↗
PubMed1996

Application of Ty1 for cloned gene insertion: amplification of a large regulated expression cassette in Saccharomyces cerevisiae.

A large cassette, 4.6 x 10(3) bases (4.6 kb) in length, containing an inducible expression system (the yeast CUP1 promoter fused to the Escherichia coli lacZ structural gene) and a bacterial neomycin-resistance gene (neo) has been cloned into the noncoding region of a GAL1-regulated Ty1 retrotransposon. Galactose was used to induce retrotransposition in Saccharomyces cerevisiae, and cells containing integrations were selected by resistance to the aminoglycoside G418. Integrations of neo and CUP1p-lacZ were verified, and beta-galactosidase activity was confirmed. Analysis via Southern blots demonstrated integrations at various chromosomal locations, and the number of insertions obtained ranged from one to five after three rounds of induction. Therefore, the packaging limit of Ty1 virus-like particles for RNA is at least 10.3 kb and Ty1 can transpose foreign genes as large as 4.6 kb, demonstrating the practical application of Ty1 for the insertion of large regulated expression cassettes.

DNA Transposable Elements↗
PubMed1994

Degenerating gypsy retrotransposons in a male fertility gene on the Y chromosome of Drosophila hydei.

During the evolution of the Y chromosome of Drosophila hydei, retrotransposons became incorporated into the lampbrush loop pairs formed by several of the male fertility genes on this chromosome. Although insertions of retrotransposons are involved in many spontaneous mutations, they do not affect the functions of these genes. We have sequenced gypsy elements that are expressed as constituents of male fertility gene Q in the lampbrush loop pair Nooses. We find that these gypsy elements are all truncated and specifically lost those sequences that may interfere with the continuity of lampbrush loop transcription. Only defective coding regions are found within the loop. Gypsy is not transcribed in loops of many other Drosophila species harboring the family. These results suggest that any contribution of gypsy to the function of male fertility gene Q does not depend on a conserved DNA sequence.

Animals↗
PubMed1996

Evolutionary origins of retroposon lineages of Mhc class II Ab alleles.

Major histocompatibility complex (Mhc) class II Ab genes have evolved into three distinct lineages. While lineage 2 alleles differ from lineage 1 alleles by the insertion of a retroposon in intron 2, the basis for the extremely large intron 2 in lineage 3 alleles has heretofore been undetermined. In this report, we demonstrate by nucleotide sequencing that the genomic sequences of prototypic alleles from all three lineages diverge significantly and that lineage 3 is derived from lineage 2 by two insertional events in intron 2. One insert, composed of a member of B1 short interspersed repetitive elements (SINEs), occurs 508 base pairs (bp) 3' of exon 2, and the other, 1141 bp 3' of exon 2 within the retroposon that distinguishes lineage 2 from lineage 1. To assess the evolutionary stability of these lineages and the extent of ancestral polymorphisms of Ab within Mus species, we extended our restriction site polymorphism analysis to include 86 alleles from 120 independently derived H2 haplotypes from 12 separate species and subspecies of Mus. A phylogenetic tree revealing the relationships of these Ab alleles with respect to restriction site polymorphisms, but excluding the retroposon insertions, demonstrated that these lineages have distinctive genomic structures beyond the retroposon polymorphisms. In summary, these mouse Ab genes were produced from successive retroposon insertion events. Lineage 1 and 2 were detected in a variety of Mus species, including Mus caroli, indicating that these lineages diverged more than 2 million years ago. Lineage 3 alleles were found only in the Mus musculus subspecies, suggesting that it diverged from lineage 2 more recently. These results indicate that all three lineages of Ab have persisted through several speciation events in the genus Mus.

Alleles↗
PubMed1994

Retrotransposon-induced ectopic expression of the Om(2D) gene causes the eye-specific Om(2D) phenotype in Drosophila ananassae.

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci, which are scattered throughout the genome. Om mutations are all semidominant, neomorphic, nonpleiotropic, and associated with the insertion of a retrotransposon, tom. We have found that the Om(2D) gene encodes a novel protein containing histidine/proline repeats, and is ubiquitously expressed during embryogenesis. The Om(2D) RNA is not detected in wild-type eye imaginal discs, but is abundantly found in the center of the eye discs of Om(2D) mutants, where excessive cell death occurs. D. melanogaster flies transformed with the Om(2D) cDNA under control of the hsp70 promoter display abnormal eye morphology when heat-shocked at the third larval instar stage. These results suggest that the Om(2D) gene is not normally expressed in the eye imaginal discs, but its ectopic expression, induced by the tom element, in the eye disc of third instar larvae results in defects in adult eye morphology.

Amino Acid Sequence↗