Biomedical subjects

T Heidmann

Publications and source records attributed to T Heidmann.

At least 37 records · Page 2Linked to original sources

PubMed1997

Functional differences between the human LINE retrotransposon and retroviral reverse transcriptases for in vivo mRNA reverse transcription.

We have analysed the reverse transcriptase (RT) activity of the human LINE retrotransposon and that of two retroviruses, using an in vivo assay within mammalian (murine and human) cells. The assay relies on transfection of the cells with expression vectors for the RT of the corresponding elements and PCR analysis of the DNA extracted 2-4 days post-transfection using primers bracketing the intronic domains of co-transfected reporter genes or of cellular genes. This assay revealed high levels of reverse-transcribed cDNA molecules, with the intron spliced out, with expression vectors for the LINE. Generation of cDNA molecules requires LINE ORF2, whereas ORF1 is dispensable. Deletion derivatives within the 3.8 kb LINE ORF2 allowed further delineation of the RT domain: > 0.7 kb at the 5'-end of the LINE ORF2 is dispensable for reverse transcription, consistent with this domain being an endonuclease-like domain, as well as 1 kb at the 3'-end, a putative RNase H domain. Conversely, the RT of the two retroviruses tested, Moloney murine leukemia virus and human immunodeficiency virus, failed to produce similar reverse transcripts. These experiments demonstrate a specific and high efficiency reverse transcription activity for the LINE RT, which applies to RNA with no sequence specificity, including those from cellular genes, and which might therefore be responsible for the endogenous activity that we previously detected within mammalian cells through the formation of pseudogene-like structures.

3T3 Cells↗

PubMed1997

Expression of intracisternal A-particle retrotransposons in primary tumors of oncogene-expressing transgenic mice.

Intracisternal A-Particle (IAP) sequences are endogenous retrovirus-like mobile elements, present at 1000 copies in the mouse genome. These elements transpose in a replicative manner via an RNA intermediate and its reverse transcription, and their transposition should therefore be tightly controlled by their transcription level. The in vivo pattern of expression of these potentially mutagenic elements had previously been analysed in normal mice, and we have now investigated their expression in transgenic mice carrying different oncogenes (e.g. c-myc, v-Ha-ras, SV40 T-antigen) under tissue-specific promoters and disclosing tumors within the brain, the mammary or salivary glands, or the lymphoid organs. Northern blot analysis of IAP expression within the resulting tumors demonstrates a lack of significant and/or systematic effect of v-Ha-ras and SV40 T-antigen expression, but a systematic IAP induction in the myc-induced tumors. In this case, however, analysis of double transgenic mice obtained by crossing the tumor-prone mice with previously described transgenic mice carrying IAP reporter genes did not provide any evidence for induction of the IAP transgenes, therefore strongly suggesting that c-myc expression had an effect on only a limited number of IAP sequences--most probably depending on their position and/or methylation state. These results strengthen the importance of in vivo studies for a correct appraisal of complex biological processes, and moderate previous conclusions derived from in vitro analyses on the general activation of IAPs by oncogenes and on the role of these transposable elements in tumorigenesis.

Animals↗

PubMed1997

Characterization of two age-induced intracisternal A-particle-related transcripts in the mouse liver. Transcriptional read-through into an open reading frame with similarities to the yeast ccr4 transcription factor.

Intracisternal A-particle (IAP) sequences are endogenous retrovirus-like elements present at 1,000 copies in the mouse genome. We had previously identified IAP-related transcripts of unusual size (6 and 10 kilobases (kb)), which are observed exclusively in the liver of the aging mouse. In this report, using cDNA libraries that we have constructed from the liver mRNAs of an aged DBA/2 mouse, we have cloned and entirely sequenced the corresponding cDNAs. Both are initiated within the 5' long terminal repeat of a type IDelta1 IAP sequence, and correspond to a read-through into a unique flanking cellular sequence containing a 966-nucleotide open reading frame, located 3' to the IAP sequence. The 6-kb IAP-related transcript corresponds to a post-transcriptional modification of the 10-kb mRNA, and is generated by a splicing event with the donor site in the IAP sequence, and the acceptor site 5' to the open reading frame. This open reading frame is located on chromosome 3, is evolutionarily conserved, and discloses significant similarity to the yeast CCR4 transcription factor at the amino acid level. The specific expression of these age-induced transcripts, which account for more than 50% of the IAP-related transcripts in the liver of old mice, is therefore entirely consistent with the induction of a single genomic locus, thus strengthening the importance of position effects for the expression of transposable elements. Characterization of this locus should now allow studies on its chromatin and methylation status, and on the "molecular factors of senescence" possibly involved in its induction.

Aging↗

PubMed1997

Cloning of a new murine endogenous retrovirus, MuERV-L, with strong similarity to the human HERV-L element and with a gag coding sequence closely related to the Fv1 restriction gene.

We had previously identified a new family of human endogenous retrovirus-like elements, the HERV-L elements (human endogenous retrovirus with leucine tRNA primer), whose pol gene was most closely related to that of the foamy viruses. HERV-L pol-related sequences were also detected in other mammalian species. The recent cloning of the mouse Fv1 gene (S. Best, P. Le Tissier, G. Towers, and J. P. Stoye, Nature 382:826-829, 1996) has shed light on another HERV-L domain--identified as a gag gene based on its location within the provirus--which was found to be 60% identical, at the nucleotide level, to the Fv1 open reading frame (ORF). We have now cloned the murine homolog of HERV-L which, in contrast to HERV-L, displays fully open reading frames in the gag and pol genes. Its predicted Gag protein shares 43% identity with the Fv1 ORF product. Moreover, the characteristic major homology region of the capsid subdomain can be identified within both proteins, thus strongly emphasizing the gag-like origin of Fv1.

Amino Acid Sequence↗

PubMed1997

Molecular cloning and characterization of a transcription factor for the copia retrotransposon with homology to the BTB-containing lola neurogenic factor.

By transfection experiments, we previously identified a 72-bp enhancer sequence within the Drosophila copia retrotransposon which is involved in the control of the transcription level of this mobile element in cells in culture. Gel shift assays with nuclear extracts from Drosophila hydei-derived DH-33 cells further demonstrated specific interactions of at least two nuclear factors with this enhancer sequence. Using this sequence as a probe for the screening of an expression cDNA library that we constructed from DH-33 cells RNA, we have isolated a cDNA clone encoding a 110-kDa protein with features common to those of known transcription factors; these include a two-zinc-finger motif at the C terminus, three glutamine-rich domains in the presumptive activation domain of the protein, and an N-terminal domain which shares homology with the Bric-à-brac, Tramtrack, and Broad-Complex BTB boxes. The precise DNA recognition sequence for this transcription factor has been determined by both gel shift assays and footprinting experiments with a recombinant protein made in bacteria. The functionality of the cloned element was demonstrated upon transcriptional activation of copia reporter genes, as well as of a minimal promoter coupled with the identified target DNA sequence, in cotransfection assays in cells in culture with an expression vector for the cloned factor. Southern blot and nucleotide sequence analyses revealed a related gene in Drosophila melanogaster (the lola gene) previously identified by a genetic approach as involved in axon growth and guidance. Transfection assays in cells in culture with lola gene expression vectors and in situ hybridization experiments with lola gene mutants finally provided evidence that the copia retrotransposon is regulated by this neurogenic gene in D.melanogaster, with a repressor effect in the central nervous systems of the embryos.

Amino Acid Sequence↗

PubMed1996

Germ line-specific expression of intracisternal A-particle retrotransposons in transgenic mice.

Intracisternal A-particle (IAP) sequences are endogenous retrovirus-like mobile elements, or retrotransposons, present at 1,000 copies in the mouse genome. These elements transpose in a replicative manner via an RNA intermediate and its reverse transcription, and their transposition should therefore be tightly controlled by their transcription level. To analyze the in vivo pattern of expression of these retrovirus-like elements, we constructed several independent transgenic mice with either a complete IAP element marked with an intron or with the IAP promoter, or long terminal repeat (LTR), alone controlling the expression of a lacZ reporter gene with a nuclear localization signal. For all transgenic lines analyzed, IAP expression as determined by reverse transcription-PCR analysis was found to be essentially restricted to the male germ line. Furthermore, in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining of all organs disclosed specific beta-galactosidase-positive blue cells only within the testis, found as patches along the seminiferous tubules and often organized as assemblies of 2, 4, 8, or 16 cells. Histochemical analyses of tissues from 13.5-day-old embryos to adults demonstrated that this LTR activity is restricted to gonocytes and premeiotic undifferentiated spermatogonia. Finally, analysis of the methylation status of both transgenes and endogenous IAP LTRs demonstrated identical patterns, with methylation in somatic tissues and hypomethylation in the testis. Transgenic mice therefore reveal an intrinsic, highly restricted IAP expression which had escaped detection in previous global Northern (RNA) blot analyses and with possible strong biological relevance, as IAP activation specifically within the germ line might be a way to generate diversity at the evolutionary level without being deleterious to individuals.

Animals↗

PubMed1995

IAP retrotransposons in the mouse liver as reporters of ageing.

IAP are endogenous retrovirus-like elements present at a thousand copies in the murine genome. They can modulate the level of expression of the tagged genes into which they have inserted, and conversely their activity could be influenced by the level of activity of the genes and/or DNA sequences into which they are embedded. In this report, we have analysed by Northern blots the pattern of expression of the IAP-related transcripts in the organs of young and ageing mice. We show that IAP transcripts of unexpected size (namely 10 kb and 6 kb) are induced in the liver of ageing mice from all inbred and hybrid strains tested. These transcripts are not detected in young mice, and their intensity disclose variations depending on the strain, as those observed for the two canonical 7.2 and 5.4 kb IAP transcripts. It is suggested that these age-dependent IAP transcripts originate from unique sites within the mouse genome that are 'tagged' by an IAP sequence, which would be sensitive both to strain-dependent cellular factors acting at the level of all IAPs, and to an age-dependent liver-specific cellular factor and/or DNA state, responsible for the position-dependent effect. These age-dependent transcripts should allow the identification of putative genes or factors of 'senescence'.

Aging↗

PubMed1995

mRNA retroposition in human cells: processed pseudogene formation.

Using a sensitive assay for detection of reverse transcription events, we demonstrate that human HeLa cells can 'retropose', i.e. reverse transcribe and integrate, the mRNA of a naive reporter gene, at a low but detectable frequency. Furthermore, we show that the retroposed copies have all the hallmarks of the processed pseudogenes naturally found in the mammalian genome: they lack intron and 5' promoter sequence, they have acquired a 3' poly(A) tail, and they are flanked by short repeats (< 15 bp) of target DNA sequence. These results demonstrate that human cells possess an endogenous reverse transcription activity, which is not restricted to transcripts of transposable elements, and which is likely to be involved in the formation, still ongoing, of a large fraction of the eukaryotic genome.

Base Sequence↗

PubMed1995

Defective I elements introduced into Drosophila as transgenes can regulate reactivity and prevent I-R hybrid dysgenesis.

The I-R hybrid dysgenesis syndrome is characterized by a high level of sterility and I element transposition, occurring in the female offspring of crosses between males of inducer (I) strains, which contain full-length transposable I elements, and females of reactive (R) strains, devoid of functional I elements. The intensity of the syndrome in the dysgenic cross is essentially dependent on the reactivity level of the R females, which is ultimately controlled by still unresolved polygenic chromosomal determinants. In the work reported here, we have introduced a transposition-defective I element with a 2.6 kb deletion within its second open reading frame into a highly reactive R strain, by P-mediated transgenesis. We demonstrate that this defective I element gradually alters the level of reactivity in the three independent transgenic lines that were obtained, over several generations. After > 15 generations, the transgenic Drosophila show strongly reduced reactivity, and finally become refractory to hybrid dysgenesis, without, however, acquiring the inducer phenotype. Induction of a low reactivity level is reversible--reactivity again increases upon transgene removal--and is maternally inherited, as observed for the control of reactivity in natural R strains. These results demonstrate that defective I elements introduced as single-copy transgenes can act as regulators of reactivity, and suggest that some of the ancestral defective pericentromeric I elements that can be found in all reactive strains could be the molecular determinants of reactivity.

Animals↗

PubMed1995

The dimerization/packaging sequence is dispensable for both the formation of high-molecular-weight RNA complexes within retroviral particles and the synthesis of proviruses of normal structure.

Retroviral particles contain a dimer of two genomic RNA molecules, linked by noncovalent intermolecular bonds. Studies by electron microscopy of viral RNA extracted from virions as well as in vitro studies have implicated a sequence, designated the dimer linkage sequence (DLS), in the dimerization process. The DLS has been localized within a short region encompassing the psi packaging sequence, between nucleotides 212 and 563 for the Moloney murine leukemia retrovirus (MoMLV) RNA. In this report, we show that viral RNAs lacking both the DLS and psi packaging sequences--and even an RNA lacking the first 6,537 nucleotides of MoMLV--can assemble within retroviral particles as high-molecular-weight, slow-migrating, heat-sensitive complexes closely related to those observed for wild-type viral RNAs. Furthermore, we show that proviruses of normal structure are generated upon infection of test cells with retroviral particles which contain the DLS/psi-deleted viral RNAs. These observations demonstrate that the DLS and psi packaging sequences are not essential in cis to form a functional RNA complex for reverse transcription and integration.

3T3 Cells↗

PubMed1995

Isolation of novel human endogenous retrovirus-like elements with foamy virus-related pol sequence.

A new class of reverse transcriptase coding sequences was detected in reverse-transcribed RNAs from human placenta by polymerase chain amplification with primers in highly conserved regions of the pol gene of mammalian retroviruses and retrotransposons. Using one of these novel sequences as a probe to screen a human genomic library, we isolated retrovirus-like elements bordered by long terminal repeats and having a potential leucine tRNA primer-binding site. Determination of the complete nucleotide sequence (6,591 bp) of one of these elements, termed HERV-L (for human endogenous retrovirus with leucine tRNA primer), revealed domains of amino acid similarities to retroviral reverse transcriptase and integrase proteins. In addition, a region with homologies to dUTPase proteins was found unexpectedly downstream from the integrase domain. Amino acid sequence and phylogenetic analysis indicate that the HERV-L pol gene is related to that of foamy retroviruses. HERV-L-related sequences are detected in several mammalian species and have expanded in primate and mouse genomes up to 100 to 200 copies.

Amino Acid Sequence↗

PubMed1994

Identification of a strong transcriptional activator for the copia retrotransposon responsible for its differential expression in Drosophila hydei and melanogaster cell lines.

We have characterized the regulatory properties of a 72bp sequence located in the 5' untranslated domain of the Drosophila copia retrotransposon, 3' to the left LTR, by transient transfection assays with cell lines derived from either Drosophila hydei (DH33 cells) or Drosophila melanogaster (Schneider II and Kc cells). Reporter plasmids were constructed which contained the lacZ gene under the control of either the entire copia LTR with 5' untranslated domain, or a minimal heterologous promoter flanked with the identified copia regulatory sequences. Upon transfection into the copia-free DH33 cells, the presence of the 72bp sequence resulted for all reporter plasmids in a 100-700 fold increase in expression level -as well as in reporter gene RNA levels- whereas this sequence had no enhancing effect upon transfection of the same plasmids into the copia-containing Schneider II or Kc cells. Moreover, mobility shift assays with the 72bp enhancer sequence disclosed two specific bands of retarded mobility with whole-cell extracts from DH33 cells, whereas no retarded band could be detected, under identical conditions, with extracts from Schneider II cells. UV crosslinking experiments between the enhancer sequence and DH33 extracts revealed a single protein species -of app. mol. wt. 50kD- for both retarded bands, thus strongly suggesting that they simply correspond to the sequential binding of two identical factor molecules to the enhancer sequence. These data demonstrate that the copia-free D. hydei cells express a strong transcriptional activator for the copia element and possible interpretations for the absence of this factor in the copia-containing D. melanogaster cells are discussed in terms of a possible "adaptation" of the "host" (D. melanogaster) to an otherwise highly mutagenic "parasite" (copia with its transcription factor).

Animals↗

PubMed1994

Retrotransposition of the Drosophila LINE I element can induce deletion in the target DNA: a simple model also accounting for the variability of the normally observed target site duplications.

Retrotransposition of the Drosophila melanogaster LINE I element normally generates target site duplications of variable length, as classically observed for most LINE elements. Using an I element "marked" with an indicator gene for in vivo detection of transposition that we previously developed, we show that deletion in the target DNA can also take place, as a direct consequence of I element transposition. We propose a simple model accounting for the generation of both target site duplications of variable length and target DNA deletions, which relies upon template switching of the LINE-encoded reverse transcriptase between single-strand DNA at the target site and the LINE template.

Animals↗

PubMed1994

Retrotransposition of a marked Drosophila line-like I element in cells in culture.

We have marked a Drosophila transposable element--the LINE-like I element--with an intron-containing indicator gene inserted in place of a large deletion in the I element second ORF encompassing the reverse transcriptase domain, and this marked element was placed downstream to a potent actin promoter. An expression vector for the I element ORFs was also constructed, under the same heterologous promoter. The indicator gene contains a lacZ reporter gene the expression of which is conditioned by retrotransposition of the marked element, thus allowing detection of transposition events by testing for either beta-galactosidase expression or occurrence of spliced DNA molecules. The marked I element was introduced into Drosophila melanogaster cells in culture by transfection. Spliced DNA copies of the marked element and specifically stained beta-galactosidase-expressing cells were detected only upon co-transfection with the I expression vector, thus indicating that an ORF2-deleted element can be complemented in trans for transposition. This simple assay for retrotransposition in Drosophila cells in culture provides a tool for the rapid analysis of the mechanism of I transposition in its cis and trans sequence requirements.

Animals↗

PubMed1993

The Drosophila copia retrotransposon contains binding sites for transcriptional regulation by homeoproteins.

We have identified in the 5' untranslated region of the Drosophila copia retrotransposon, 3' to the left LTR, a sequence for transcriptional regulation by homeoproteins. Co-transfection assays using expression vectors for homeoproteins and reporter vectors containing the lacZ gene under the control of either the entire copia LTR with 5' untranslated sequence, or a minimal heterologous promoter flanked with a 130 bp fragment containing the copia untranslated region, disclosed both positive and negative modulations of promoter activity in Drosophila cells in culture: a 5-10 fold decrease with engrailed, even-skipped and zerknüllt in DH33 cells, and a 10-30 fold increase with fushi tarazu and zerknüllt in Schneider II cells. In all cases, the regulatory effects were abolished with reporter plasmids deleted for a 58 bp fragment encompassing the putative homeoprotein binding sites. Mobility shift assays with a purified homeodomain-containing peptide demonstrated direct interaction with the 58 bp fragment, with an affinity in the 1-10 nM range as reported with the same peptide for other well characterized homeodomain binding regulatory sites. Foot-printing experiments with the extended LTR demonstrated protection of 'consensus' sequences, located within the 58 bp fragment. These homeodomain binding sites could be involved in the developmental regulation of the copia retrotransposon.

Animals↗

PubMed1993

Generation of processed pseudogenes in murine cells.

Using as a reporter gene a non-coding proviral structure marked with an intron-containing indicator, we demonstrate the de novo formation, via a retrotransposition pathway, of canonical processed pseudogenes in cultured mammalian cells. Their structural features include endings corresponding to the start and termination of the RNA intermediate, intron loss, acquisition of a 3' poly(A) tail, and target site duplications of variable length. The absence of extracellular intermediates for these processes, and the elimination during retrotransposition of sequences in the reporter gene essential in cis for a retroviral cycle, further suggest that endogenous retroviruses or related elements are not involved. Pseudogene formation frequency is markedly increased (up to 10-fold) by several treatments including treatment with 5-azacytidine or tetradecanoyl phorbol acetate, or serum starvation, which do not act at the reporter gene transcription level, but rather on endogenous genes--including the LINE elements--necessarily involved in trans-complementation for retrotransposition.

3T3 Cells↗

PubMed1992

High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay.

We devised an indicator gene for retrotransposition, nlsLacZRT, which contains the Escherichia coli lacZ gene fused to a nuclear location signal (nlsLacZ), engineered in such a way that the gene is expressed only if the structure in which it has been inserted transposes itself through an RNA intermediate. A cloned murine leukemia retrovirus with an ecotropic host range (Moloney murine leukemia virus), rendered defective by a large deletion encompassing the three viral gag, pol, and env open reading frames, was marked with this indicator gene and introduced by transfection into heterologous feline cells. No beta-galactosidase activity could be detected among the clonal cell population, unless the defective provirus was complemented in trans by the gag-pol gene products. Under these conditions, cell variants which disclosed an easily detectable nuclear blue coloration upon in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining were observed. Fluorescence-activated cell sorting of the beta-galactosidase-positive cells, followed by Southern blot analysis, demonstrated an unambiguous correlation between nlsLacZRT activation and retrotransposition of the marked provirus. Transposition occurs at a high frequency (up to 10(-4) events per cell per generation), which is dependent on the level of expression of the gag-pol gene and is concomitant with the release of noninfectious retroviruslike particles which are the hallmarks, but not the intermediates, of the intracellular transposition process.

Animals↗

PubMed1991

Evidence for a reverse transcription intermediate for a marked line transposon in tumoral rat cells.

We have marked a "reconstituted" LINE element from rat with an intron-containing indicator gene, to test for its RNA-mediated transposition in tumoral rat cells in culture. Three cloned LINE promoter-containing fragments were tested by a transient transfection assay using a LacZ reporter gene, and the promoter with maximum expression was substituted--in an homologous manner--to the 5' domain of a close to full-length genomic LINE. The resulting marked LINE was stably introduced by transfection into tumoral rat cells. PCR amplification of the DNA from several transfected clones, using primers bracketting the intronic domain of the indicator gene, yielded fragments with a reduced size: their DNA sequencing, in four cases out of four, demonstrated splicing out of the intron as expected for the passage of the marked LINE through an RNA intermediate and its reverse transcription. Fractionation of cellular DNA by the Hirt procedure indicated that reverse transcribed copies are present in the "extrachromosomal" fraction. Their abundance is close to 1 copy per 10(4) cells. These results strongly suggest that rat LINEs transpose through an RNA intermediate and its reverse transcription, as previously demonstrated for the Drosophila LINE I element and, further, that reverse transcription might take place prior to integration, resulting in extrachromosomal DNA molecules as preintegrative transposition intermediates.

Animals↗