Mouse Intracisternal A-particle Research Articles

Phylogenetic and DNA methylation analysis reveal novel regions of variable methylation in the mouse IAP class of transposons

BackgroundSelect retrotransposons in the long terminal repeat (LTR) class exhibit interindividual variation in DNA methylation that is altered by developmental environmental exposures. Yet, neither the full extent of variability at these “metastable epialleles,” nor the phylogenetic relationship underlying variable elements is well understood. The murine metastable epialleles, Avy and CabpIAP, result from independent insertions of an intracisternal A particle (IAP) mobile element, and exhibit remarkably similar sequence identity (98.5%).ResultsUtilizing the C57BL/6 genome we identified 10802 IAP LTRs overall and a subset of 1388 in a family that includes Avy and CabpIAP. Phylogenetic analysis revealed two duplication and divergence events subdividing this family into three clades. To characterize interindividual variation across clades, liver DNA from 17 isogenic mice was subjected to combined bisulfite and restriction analysis (CoBRA) for 21 separate LTR transposons (7 per clade). The lowest and highest mean methylation values were 59% and 88% respectively, while methylation levels at individual LTRs varied widely, ranging from 9% to 34%. The clade with the most conserved elements had significantly higher mean methylation across LTRs than either of the two diverged clades (p = 0.040 and p = 0.017). Within each mouse, average methylation across all LTRs was not significantly different (71%-74%, p > 0.99).ConclusionsCombined phylogenetic and DNA methylation analysis allows for the identification of novel regions of variable methylation. This approach increases the number of known metastable epialleles in the mouse, which can serve as biomarkers for environmental modifications to the epigenome.

Proteinases of betaretroviruses bind single-stranded nucleic acids through a novel interaction module, the G-patch

Retroviral proteinases (PRs) are essential for retrovirus infectivity but the mechanism of their activity regulation is poorly understood. We investigated possible involvement in this process of the C-terminal domain (CTD) of betaretroviral PRs. We found that the presence of CTD attenuates proteolytic activity of Mason-Pfizer monkey virus PR, while it does not significantly affect the activity of mouse intracisternal A-particle retrovirus PR. However, both PRs bind single-stranded nucleic acids through their CTDs that contain a novel binding motif, the G-patch, whose function is dependent on a single conserved tyrosine residue. Oligonucleotide binding to both PRs does not inhibit their proteolytic activity.

Mapping of mouse intracisternal A-particle proviral markers in an interspecific backcross.

We present a linkage map of intracisternal A-particle (IAP) proviral loci. The IAP family consists of 2000 endogenous proviral elements that are widely dispersed in the mouse genome. The map was constructed by using an interspecific backcross and markers defined by oligonucleotide probes specific for subclasses of expressed IAP elements. In genomic DNA from C57BL/6J mouse, these probes each detected from 12 to 44 HindIII restriction fragments that represent junctions between proviral and 5'-flanking DNA. The fragments have characteristic strain distribution patterns (SDPs) that are particularly polymorphic in the DNAs of C57BL/6J and Mus spretus mice used for the backcross. IAP loci were placed on the map by comparison of their distribution patterns with those of known genetic markers in the backcross. The map includes 51 IAP loci that have not been previously mapped and 23 IAP proviruses that had been previously mapped in recombinant inbred (RI) strains. Comparable map positions were obtained with the IAP markers in the interspecific backcross and the RI strains. The mapped IAP loci were widely dispersed on the X Chromosome (Chr) and all of the autosomes except Chrs 9 and 19, providing useful genetic markers for linkage studies.

Characterization of Murine Middle Repetitive DNA

We have characterized four sequences from a small library containing a subset of the repetitive families of the mouse. Each clone has a repetition frequency of less than 1,100 copies per genome and each clone represents a unique family of middle repetitive DNA. One clone (pMR111) shares homology with mouse intracisternal A-particle (IAP) elements, a second clone (pMR89) has partial homology with a sequence in the 3' untranslated region of the human fibulin gene, while two clones (pMR6, pMR66) are new that have no homology to any reported DNA sequence. Each clone hybridizes to one or two discrete RNA transcripts from one or more tissues of the mouse. Clone pMR66 detected restriction fragment length polymorphisms (RFLPs) at three loci in genomic mouse DNA, defining loci at the distal end of chromosome 5 and the proximal end of chromosome 7. The third locus is unmapped. This study demonstrates that cloned repeats from a repetitive DNA library are a potential source of genetic markers.

Molecular cloning and characterization of a complete Chinese hamster provirus related to intracisternal A particle genomes

We report here the nucleotide sequence of a full-length Chinese hamster genomic proviral element, CHIAP34. CHIAP34 is 6,403 bp long with long terminal repeats of 311 bp at each end. The genetic organization of CHIAP34 was determined by comparison with intracisternal A particle (IAP) genetic elements from the mouse and Syrian hamster. Extensive homology at the nucleotide and deduced amino acid sequence levels was observed between CHIAP34 and the mouse and Syrian hamster IAP elements. CHIAP34 may represent a defective Chinese hamster IAP genetic element. The gag gene consists of 837 codons, of which 558 codons are in a single long open reading frame followed by several frameshifts. The pol gene begins with a -1 frameshift and consists of a long open reading frame of 753 codons followed by a short open reading frame of 103 codons. The putative env region contains multiple termination codons in all reading frames. CHIAP34 is representative of the predominant retroviral elements in the Chinese hamster ovary cell genome present at around 80 copies per haploid genome.

Identification of a novel murine IAP-promoted placenta-expressed gene

We have cloned and characterized a novel cellular gene that is promoted by an intracisternal A-particle (IAP) LTR and expressed in the mouse placenta (mouse IAP promoted placental gene, MIPP). A 1067bp cDNA clone containing an IAP LTR U5 region duplicated in its 5' terminus and an ORF coding for a potential 202 amino acids protein was isolated from an 8.5 day old mouse embryo cDNA library. Sequence analysis of the 5' region of a genomic clone revealed the presence of a solo IAP LTR with the same U5 duplication, and primer extension analysis confirmed that transcription of the MIPP gene is under the control of the IAP LTR. Expression of the MIPP gene parallels that of IAP genes in normal mouse tissues with abundant transcripts present in the placenta and also in the myeloma MOPC-315. The MIPP-encoded protein is composed of four 48-amino acid repeat units and shares homology with a vaccinia virus gene product. MIPP-related sequences were also detected in higher eukaryotic genomes including human.

A variant binding sequence for transcription factor EBP-80 confers increased promoter activity on a retroviral long terminal repeat.

The cloned long terminal repeats (LTRs) of mouse intracisternal A-particle (IAP) proviral elements differ in their promoter activity. In this study, the LTR from a recently transposed IAP element (rc-mos) is shown to be a more effective promoter both in vivo and in vitro than the LTR from a randomly cloned genomic element (MIA14). These LTRs differ in nucleotide sequence in certain previously defined protein-binding domains. In particular, the MIA14 LTR contains two domains, designated Enh1 and Enh2, with sequence homology to the SV40 enhancer core motif, while in rc-mos the Enh2 position is occupied by a variant sequence which lacks core homology. EBP-80 is a general enhancer core-binding protein originally isolated by virtue of its affinity for the MIA14 Enh2 sequence (Falzon, M., and Kuff, E.L. (1989) J. Biol. Chem. 264, 21915-21922). We now find by quantitative binding studies, binding competition, and UV cross-linking that EBP-80 from both human and mouse cells binds to the "Enh2" motif of rc-mos more strongly than to the Enh2 of MIA14. In vitro transcription from both LTRs is strongly enhanced by addition of EBP-80 showing that binding is related to function. The rc-mos LTR remains the more effective promoter in the presence of added EBP-80. Reciprocal substitution of the Enh2 domains in the two LTRs by site-directed mutagenesis shows that the rc-mos variant confers a 3-fold increment in in vivo promotor activity. The rc-mos motif or a closely related sequence is found in the cloned LTRs of many expressed and/or recently transposed IAP elements. EBP-80 is identified as a cellular transcription factor whose heightened levels in certain mouse cells might result in preferential expression of IAP elements containing this sequence motif.

Isolation and Characterization of a Protein Fraction That Binds to Enhancer Core Sequences in Intracisternal A-particle Long Terminal Repeats

The U3 region of mouse intracisternal A-particle (IAP) long terminal repeats (LTRs) contains several nuclear protein-binding domains. Two of these contain sequences with homology to the SV40 enhancer core. We refer to these two domains as Enh1 and Enh2. The Enh2 domain is an important determinant of promoter activity in vivo. We report here the isolation of nuclear fractions from human 293 and mouse MOPC-315 cells which interact with Enh1 and Enh2. Purification was achieved via DNA-affinity chromatography on a multimerized oligonucleotide representing the Enh2 region from the LTR of the mouse genomic IAP element, MIA14. Glycerol gradient sedimentation suggested a native Mr of approximately 80-100 for the binding component(s) in both crude and affinity-purified fractions. UV cross-linking showed that the binding activity involved two polypeptides within this size range. The affinity-isolated fraction from each cell line was highly purified, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in vitro binding analysis. Exonuclease III footprinting showed that the two polypeptides interacted preferentially with the Enh1 and Enh2 domains within a 139-base pair segment from the MIA14 LTR. The polypeptides interacted in a sequence-specific manner with oligonucleotides representing these domains within the IAP LTR and with oligonucleotides containing the enhancer core sequence from SV40 and polyoma virus. Equilibrium binding studies indicated that the apparent dissociation constants for the polypeptides binding to the enhancer core sequence from MIA14, SV40, and polyoma virus were similar. Therefore, this affinity-purified fraction may represent a novel enhancer core-binding component which is distinct from the previously characterized rat CCAAT/enhancer-binding protein, C/EBP.

Multiple protein-binding sites in an intracisternal A particle long terminal repeat.

The long terminal repeats (LTRs) of cloned intracisternal A particles (IAPs) can function as effective promoters in heterologous and homologous cell types (K. K. Lueders, J. W. Fewell, E. L. Kuff, and T. Koch, Mol. Cell. Biol. 4:2128-2135, 1984) and respond to transcriptional factors induced by various nuclear oncogene products (S. Luria and M. Horowitz, J. Virol. 57:998-1003, 1986). Using the first 139 base pairs of the U3 region of a cloned mouse IAP LTR as probe, we demonstrated multiple exonuclease III stop sites which appeared specifically in the presence of nuclear extract protein. Various extracts gave similar footprints, but the amount of nuclear protein required varied up to 10-fold. Cell lines transformed with known nuclear oncogenes, such as adenovirus E1a and E1b (293 cells), simian virus 40 large T antigen (COS7 cells), and c-myc (MOPC-315 cells) had more and/or higher-affinity factors for the IAP LTR than extracts from HeLa, CV1, and NIH 3T3 cells did. DNase I footprinting revealed at least five distinct protein-binding domains within the 139-base-pair region. These domains correspond to segments of highly conserved nucleotide sequence among a number of IAP LTRs. Gel retardation studies with oligonucleotides encompassing the DNase I footprint sites showed that the nuclear factors are present in different proportions and different absolute levels in extracts from different cell types. Moreover, the oligonucleotide probes indicate that individual motifs can be occupied independently of one another. Three of the DNase I footprints include a sequence with homology to the simian virus 40 core enhancer and sequence motifs that closely resemble the binding sites for transcription factors SP1 and AP-1. The other two binding sites are not obviously related to previously recognized motifs. The multiple protein-binding sites in close proximity indicate the complex regulatory mechanism for IAP transcription.

Nucleotide sequence of a complete mouse intracisternal A-particle genome: relationship to known aspects of particle assembly and function.

The 7,095-nucleotide sequence of a mouse genomic intracisternal A-particle (IAP) element, MIA14, is reported. MIA14 is known to be colinear with IAP 35S RNA and to contain functional long terminal repeats. Its internal genetic organization was determined by comparisons with a homologous Syrian hamster element and the related retroviruses simian retrovirus 1 (simian type D) and Rous sarcoma virus (avian type C). MIA14 contains a gag-protease open reading frame of 827 codons and a pol region of 867 codons entered by a frame shift of -1. The env region of 1,100 base pairs has multiple stop codons in all reading frames, consistent with the failure thus far to detect IAP-related glycosylated envelope components. RNA transcribed in vitro from a cDNA clone containing a closely homologous gag-protease open reading frame was translated in a cell-free system. The main product was a 73-kilodalton polypeptide immunoprecipitable with antiserum against the authentic IAP gag-related structural protein p73. Rather than ending at the gag-protease boundary, p73 appears to contain 7 to 8 kilodaltons of peptide encoded by the protease domain, a peculiarity possibly related to the observed impairment of normal protein processing in IAPs. The N-terminal 217 codons of gag are unique to murine IAPs and may have been contributed by recombination with a cellular gene. The mouse-specific region of gag encodes a hydrophobic signal peptide with an atypical cleavage site. Delayed cleavage of this peptide could result in anchoring of newly synthesized p73 to the endoplasmic reticulum membrane and restriction of particle assembly to this site.

MRNA from human colon tumor and mucosa related to the pol gene of an endogenous A-type retrovirus

The expression of endogenous retroviral genes in mammals may be etiologically related to genetic diseases including cancer. Recently, A-type human endogenous retroviral genomes have been cloned using the reverse transcriptase (pol) genes of rodent intracisternal A-particles (IAP). In this report, RNA from human colon adenocarcinoma and surrounding mucosa was hybridized to mouse IAP pol and gag genes to examine the expression of human endogenous A-type retroviruses. Abundant, heterogeneous size, polyadenylated transcripts homologous to the mouse IAP pol gene were detected in both tissues. Transcripts homologous to the mouse IAP gag region were not found.

In vitromethylation inhibits the promotor activity of a cloned intracisternal A-particle LTR

We studied the relation between LTR methylation and expression of the family of endogenous retrovirus-like elements related to mouse intracisternal A-particles (IAP). Comparative HpaII/MspI and HhaI restriction analysis of genomic DNA's showed that in cells and tissues with a low level of IAP gene expression, HpaII and HhaI sites within the 5' LTR were heavily methylated, while in cells abundantly expressing IAP's 20 to 30% of the 5' LTRs were demethylated at these sites. The effects of methylation on the promoter activity of a cloned IAP 5' LTR was studied directly, using the plasmid pMIA5' L-cat in which this LTR was linked to the chloramphenicol acetyl transferase (CAT) gene. In vitro methylation of three HhaI sites located between -137 and -205 bp from the RNA start site of this LTR completely inactivated the promoter activity of pMIA5' L-cat transfected into COS7 cells. Methylation of a HpaII site located 94 bp downstream from the RNA start site reduced the promoter activity by 75%. The results show that methylation at sites both upstream and downstream from the RNA start site profoundly effects the promoter activity of this LTR and suggest that methylation within the 5' LTR can serve to regulate IAP gene expression in vivo.

Structural analysis of type II variants within the mouse intracisternal A-particle sequence family

Intracisternal A-particle (IAP) elements are present in multiple copies in the mouse and other rodent genomes. The bulk of this sequence family in Mus musculus consists of 7 Kb long elements, but the majority of IAP sequences involved in known transpositions have been deleted forms. The present study describes a subset of deleted IAP sequences (type II IAP) characterized by insertion of a particular short sequence element (AIIins). AIIins are interspersed and the majority occur as part of the type II IAP elements in the mouse genome. AIIins sequences are absent or in low copy number outside Mus musculus. We have isolated clones containing AIIins from a mouse genomic DNA library and have sequenced three isolates of AIIins and their surrounding IAP sequences to define the detailed structure of type II elements. AIIins are 272, 268 and 264 bp long and 90% homologous in sequence. They are bracketed by 9 bp duplications, suggesting they may be inserted elements. A 75 bp region containing a core enhancer sequence is repeated at the 5' end in type II IAP elements. Insertion into the IAP genome, with potential to encode an integrase function, may have played a role in the amplification of AIIins.

Reverse transcriptase of rat liver associated with the endogenous retrovirus related to the mouse intracisternal A-particles

Reverse transcriptase activity was found in rat liver enclosed in virus-like particles. Through hybridization with DNA probes of A- and C-type retroviruses and with the help of electron microscopy the virus-like particles have been identified as endogenous retroviruses related to the mouse intracisternal A-particles. Blot hybridization revealed the provirus DNA in the rat genome. The enzyme was isolated from the virus-like particles, purified and characterized. The main properties of the enzyme resemble those of the mammalian retrovirus reverse transcriptase.

The long terminal repeat of an endogenous intracisternal A-particle gene functions as a promoter when introduced into eucaryotic cells by transfection.

We describe experiments designed to determine whether an endogenous intracisternal A-particle (IAP) gene randomly selected from a mouse embryo library has the potential to be transcriptionally active. Assays for IAP gene transcription were done with permanently transformed rat cells and transiently transfected monkey and mouse cells. The rat cells, which had integrated IAP gene copies, contained IAP RNA. A start site within the IAP 5' long terminal repeat (LTR) was localized by S1 mapping. The promoter activity of the IAP LTR was also measured in cells 48 h after the introduction of recombinant plasmids in which bacterial chloramphenicol acetyl transferase (CAT) encoding sequences were under the control of the LTR. The IAP LTR promoted CAT activity in mouse and monkey cells. In mouse L-cells, the levels of CAT activity were 10 to 25% of those promoted by an analogous recombinant containing the Moloney murine sarcoma virus LTR as the promoter. In contrast to the Moloney murine sarcoma virus LTR, the IAP LTR was five- to eightfold more active in monkey cells than in mouse cells. The 5' and 3' LTRs were equally active, and promoter activity was dependent on having the orientation of the LTRs with respect to the CAT gene the same as their orientation with respect to the IAP gene. A 5'-flanking sequence containing a member of the highly repetitive R-sequence family increased CAT activity in COS cells 11-fold when present along with the LTR. Our results indicate that the LTR of an endogenous mouse IAP gene can function as an efficient promoter in heterologous as well as homologous cells.

The long terminal repeat of an endogenous intracisternal A-particle gene functions as a promoter when introduced into eucaryotic cells by transfection.

We describe experiments designed to determine whether an endogenous intracisternal A-particle (IAP) gene randomly selected from a mouse embryo library has the potential to be transcriptionally active. Assays for IAP gene transcription were done with permanently transformed rat cells and transiently transfected monkey and mouse cells. The rat cells, which had integrated IAP gene copies, contained IAP RNA. A start site within the IAP 5' long terminal repeat (LTR) was localized by S1 mapping. The promoter activity of the IAP LTR was also measured in cells 48 h after the introduction of recombinant plasmids in which bacterial chloramphenicol acetyl transferase (CAT) encoding sequences were under the control of the LTR. The IAP LTR promoted CAT activity in mouse and monkey cells. In mouse L-cells, the levels of CAT activity were 10 to 25% of those promoted by an analogous recombinant containing the Moloney murine sarcoma virus LTR as the promoter. In contrast to the Moloney murine sarcoma virus LTR, the IAP LTR was five- to eightfold more active in monkey cells than in mouse cells. The 5' and 3' LTRs were equally active, and promoter activity was dependent on having the orientation of the LTRs with respect to the CAT gene the same as their orientation with respect to the IAP gene. A 5'-flanking sequence containing a member of the highly repetitive R-sequence family increased CAT activity in COS cells 11-fold when present along with the LTR. Our results indicate that the LTR of an endogenous mouse IAP gene can function as an efficient promoter in heterologous as well as homologous cells.