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Results for “Genes, Intracisternal A-Particle”

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PubMed1991

Binding of the transcription factor EBP-80 mediates the methylation response of an intracisternal A-particle long terminal repeat promoter.

Intracisternal A-particle (IAP) expression in mouse cells has been correlated with hypomethylation of HhaI and HpaII sites in proviral long terminal repeats (LTRs). In a previous study, in vitro methylation of three HhaI sites in the U3 region of the LTR from the cloned genomic IAP element, MIA14, was shown to inhibit promoter activity in vivo. In this study, we found by site-directed mutagenesis that the two more downstream HhaI sites within this LTR were responsible for the methylation effects on promoter activity in vivo; methylation of the other (5') HhaI site, which lies within a putative SP1 binding domain, did not affect promoter activity. Methylation of the HhaI sites also inhibited promoter activity of the LTR in a cell-free transcription system. Exonuclease III footprinting demonstrated methylation-induced changes in protein binding over the region encompassing the downstream HhaI site, designated the Enh2 domain. The protein that interacts specifically with this domain, EBP-80, was characterized in a previous study (M. Falzon and E. L. Kuff, J. Biol. Chem. 264:21915-21922, 1989). We show here that the presence of methylcytosine in the HhaI site within the Enh2 domain inhibited binding of EBP-80 in vitro. The methylated MIA14 LTR construct was much less responsive to added EBP-80 in an in vitro transcription system than was the unmethylated construct. These data suggest that CpG methylation within the Enh2 domain may exert its effect on transcription in vivo by altering the interaction between EBP-80 and its cognate DNA sequence.

Base Sequence↗

PubMed1992

Interaction of SEWA sarcoma cell proteins with the intracisternal A-type particle long terminal repeat DNA sequence.

Intracisternal A-type particle (IAP) transcripts are endogenous retrovirus-like sequences expressed during specific stages of normal development and in a variety of murine tumors. In this study, we have analyzed two cell lines derived originally from the SEWA murine osteosarcoma and grown either as ascites or as solid tumors, for proteins that might regulate IAP expression. We found that subline AA7-NA, originally derived from the ascites tumor, expressed about five times more IAP RNA than the AS12-AD subline, which was derived from a solid tumor. In view of this finding, we examined the binding of cellular proteins from the two cell lines to the 5' end of an IAP long terminal repeat sequence. Gel retardation assays of DNA-protein complexes and DNase I footprinting assays identified several DNA sequences within the long terminal repeat fragment that were protected by protein extracts from both SEWA sublines. Gel retardation assays using specific synthetic oligonucleotide sequences that correspond to two of these protected regions revealed different patterns of DNA-protein complexes with extracts from the two SEWA sublines. These data suggest that expression of IAP sequences is regulated by complex mechanisms involving several proteins that appear to differ between the two sublines.

Animals↗

PubMed1991

Integration site preferences of endogenous retroviruses.

Retroviruses have the ability to integrate into the genome of their host, in many cases with little apparent sequence or site specificity. However, relatively few studies have addressed more general features of chromosomal integration. In this study we directly visualized the chromosomal organization of three representative endogenous retroviruses by in situ hybridization. Because there are 50-1000 copies of each of these retroviruses in the genome, it was possible to evaluate repeated integration events. Each retroviral sequence exhibited a unique and markedly different integration pattern. In order to characterize more precisely the chromosomal domains targeted by each retrovirus, later replicating domains were differentially labeled. Additionally, prototypic SINES and LINES (short and long interspersed reiterated sequences), which are inhomogeneously distributed on chromosome arms, were simultaneously detected. Retroviral copies of greater than or equal to 2 kb were found (i) exclusively in a discrete set of later replicating domains, most of which have the staining characteristics of constitutive heterochromatin, (ii) widely represented in disparate types of chromosome domains, or (iii) almost completely confined to CpG Alu-rich regions that are known to be early replicating. Retroviral elements in Alu-rich domains would be expected to be actively transcribed in all cells. Surprisingly, hybridization to blots of brain RNA showed an approximately 25 fold lower level of transcripts from these Alu associated elements than from retroviral sequences restricted to later replicating, heterochromatic domains. Retroviral insertions may subvert more typical transcriptional characteristics of a domain. The present results indicate that there are highly specific integration patterns for each endogenous retrovirus that do not readily relate to their sequence or particle classification. Each host genome may utilize these elements for contrary, and possibly beneficial functions.

Adult↗

PubMed1991

Cell lineage-specific expression of the MIPP gene.

The expression of the mouse IAP promoted placental (MIPP) gene was examined in the tissues of the 13.5 day mouse conceptus by Northern hybridization analysis. MIPP transcripts were found in total cellular RNA isolated from the placenta, parietal yolk sac, and visceral yolk sac, but not the embryo or amnion. Expression in the visceral yolk sac was confined to the visceral endoderm cell layer suggesting that the MIPP gene is specifically expressed in tissues derived from the trophectoderm and primitive endoderm cell lineages. The expression of IAP genes paralleled that of MIPP in all tissues studied except for the presence of IAP transcripts in the amnion.

Amnion↗

PubMed1991

The expression of intracisternal A particle genes in the preimplantation mouse embryo.

Intracisternal A particles (IAP), murine endogenous retrovirus, make up 0.3% of the mouse genome. They are expressed in some normal tissues, certain transformed cell lines, and show stage-specific patterns of expression in early embryos. We have used peptide-specific antisera and the polymerase chain reaction to explore type-specific expression of these IAP during preimplantation development. In this paper we show that the IAP core protein, p73, characteristic of type IIAP, is present throughout preimplantation development while the gag-pol fusion protein p120, characteristic of the variant type I delta 1, is synthesized and expressed only from the 8-cell stage onward. Type IIAP RNA is present at all stages and appearance of p120 at the 8-cell stage could represent new transcription or translation from a preexisting I delta 1 message. The presence of type II IAP RNA varies according to stage, with two sizes of type II transcripts present at all stages except the 2-cell stage at which time only the smaller of the two transcripts can be detected. The reappearance of the larger type II transcript subsequent to the 2-cell stage implies new transcription of this type II subspecies. The presence of type I, II, and p73 in the unfertilized egg strongly suggests maternal inheritance from the oocyte.

Amino Acid Sequence↗

PubMed1991

Retrotransposition of a mouse IAP sequence tagged with an indicator gene.

We have marked a cloned mouse IAP sequence with a neomycin-containing indicator gene whose expression is conditioned by passage of the transposon through an RNA intermediate. Transposition of the marked IAP introduced into tumor cells could be detected by simple selection of the cells in G418, at a frequency of 10(-6) per cell per generation. Southern blot analysis and nucleotide sequencing after PCR amplification demonstrated "retrotransposition" of the marked element, with splicing out of an intron contained in the indicator gene, and retroviral-like reverse transcription and integration of the transposed IAPs, with 6 bp duplications of the identified target sites. Transposition was found to be mutagenic for the element, as might be expected if the identified marked and endogenous IAP transcripts were coencapsidated into IAP particles as dimers.

Animals↗

PubMed1992

Aldolase-DNA interactions in a SEWA cell system.

In this report we demonstrate the novel finding that aldolase A interacts with DNA sequences in mouse SEWA sarcoma cells. This interaction was initially observed through the identification of a 40 kDa protein which was eluted from a DNA affinity chromatography column consisting of the long terminal repeat (LTR) of the endogenous intracisternal A-type particle (IAP). Microsequencing analysis identified this 40 kDa protein as the glycolytic enzyme, aldolase A. The use of specific anti-aldolase antibodies enabled the identification and subsequent purification of aldolase from the nuclear protein fraction of two SEWA sublines, one that is adherent and one that grows in suspension. In order to confirm our initial finding that aldolase is capable of interacting with DNA, proteins from each subline were immunopurified with anti-aldolase antibodies, eluted and then tested for their ability to interact with IAP-LTR DNA sequences. Interestingly, only aldolase derived from the anchorage dependent SEWA cells was capable of interacting with the IAP-LTR, however, several cell lines derived from human tumors also exhibited this activity. Subsequent studies revealed the ability of aldolase to interact with some but not every DNA sequence tested, implying that there may be a minimal DNA conformation and/or sequence requirement for this activity. The presence of aldolase A in the nuclei and its ability to interact with certain DNA sequences suggest a novel role for this metabolic enzyme.

Animals↗

PubMed1992

Detection of a human intracisternal retroviral particle associated with CD4+ T-cell deficiency.

A number of non-human-immunodeficiency-virus (HIV) type 1 disorders are associated with CD4+ T-cell deficiency and dysfunction. However, the etiopathogenesis of CD4+ T-cell immunodeficiency in these disease states remains unclear. Human intracisternal retroviral (HICRV) particles were detected in a lymphoblastoid cell line exposed to mononuclear cells from a patient with severe CD4+ T-cell deficiency without risk factors for HIV infection. Ultrastructurally, the HICRV is distinct from HIV-1, HIV-2, human T-lymphotropic virus (HTLV) type I, and HTLV-II. Supernatants of activated mononuclear cells showed significant reverse transcriptase activity that was predominantly Mn2+ dependent. The patient's mononuclear cells were negative for HIV-1, HIV-2, HTLV-I, and HTLV-II proviruses as demonstrated by the lack of amplification by PCR. Also, the patient's serum was negative for antibodies to HIV-1, HTLV-I, and HTLV-II and for HIV-1 p24 antigen; however, serum was positive for antibodies against the HICRV as demonstrated by Western blot. Similar HICRV particles were detected in a lymphoblastoid cell line exposed to mononuclear cells from the patient's daughter, who showed CD4+ T-cell dysfunction. The HICRV may be associated with CD4+ T-cell immunodeficiency and dysfunction in patients without risk for HIV-1, HIV-2, HTLV-I, and HTLV-II.

Acquired Immunodeficiency Syndrome↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1991

The oncogenic potential of an activated Hox-2.4 homeobox gene in mouse fibroblasts.

The homeobox gene Hox-2.4 is transcriptionally activated in cells of the mouse myeloid leukemia WEHI-3B. The constitutive Hox-2.4 expression in WEHI-3B cells is due to insertion of a transposable element belonging to the family of intracisternal A particles. In this study, we demonstrated the oncogenic potential of this activated homeobox gene. NIH 3T3 fibroblast clones bearing the activated Hox-2.4 gene produced fibrosarcomas in nude mice.

Animals↗

PubMed2009

Multipotent genetic suppression of retrotransposon-induced mutations by Nxf1 through fine-tuning of alternative splicing.

Cellular gene expression machinery has coevolved with molecular parasites, such as viruses and transposons, which rely on host cells for their expression and reproduction. We previously reported that a wild-derived allele of mouse Nxf1 (Tap), a key component of the host mRNA nuclear export machinery, suppresses two endogenous retrovirus-induced mutations and shows suggestive evidence of positive selection. Here we show that Nxf1(CAST) suppresses a specific and frequent class of intracisternal A particle (IAP)-induced mutations, including Ap3d1(mh2J), a model for Hermansky-Pudlak syndrome, and Atcay(hes), an orthologous gene model for Cayman ataxia, among others. The molecular phenotype of suppression includes approximately two-fold increase in the level of correctly-spliced mRNA and a decrease in mutant-specific, alternatively-processed RNA accumulating from the inserted allele. Insertional mutations involving ETn and LINE elements are not suppressed, demonstrating a high degree of specificity to this suppression mechanism. These results implicate Nxf1 in some instances of pre-mRNA processing, demonstrate the useful range of Nxf1(CAST) alleles for manipulating existing mouse models of disease, and specifically imply a low functional threshold for therapeutic benefit in Cayman ataxia.

Alternative Splicing↗

PubMed1992

Nuclear protein binding to the 5' enhancer region of the intracisternal A particle long terminal repeat.

The interactions of nuclear proteins from embryonal carcinoma cells (PCC3) with the long terminal repeats (LTRs) of murine intracisternal A particle (IAP) genes were studied. Two protein-DNA complexes were detected between PCC3 nuclear extract and IAP LTRs in a gel mobility shift assay. An additional complex was observed when enriched fractions from a heparin-agarose column were used as the source of proteins. Two regions within the LTR of IAP 81 were identified as the sites of protein interaction by DNase I protection. One region encompasses 43 nucleotides within the U3 region at the 5' end of LTR 81. The other covers a 78 base pair region lying within 100 nucleotides upstream from the transcription initiation site. Studies using constructs containing intact or deleted versions of the LTR fused to the bacterial chloramphenicol acetyltransferase gene indicated that the absence of the 5' 47 base pairs reduced the level of chloramphenicol acetyltransferase transcription to 20% of that driven by the entire LTR. Southwestern analysis of PCC3 nuclear extracts and column fractions revealed that a 28,000- and a 46,000-dalton protein were the major species that interact with the 5' end of IAP LTR 81.

Animals↗

PubMed1992

Insertion of an intracisternal A particle within the 5'-regulatory region of a gene encoding folate-binding protein in L1210 leukemia cells in response to low folate selection. Association with increased protein expression.

We have previously described a subline of L1210 murine leukemia cells (LL1) selected in a low level of 5-formyltetrahydrofolate which overexpresses a membrane-bound folate-binding protein (FBP1) and exhibits a rearrangement at the locus encoding this protein. Genomic clones containing the entire FBP1-encoding DNA from both L1210 and LL1 were isolated and characterized. Sequence analysis indicates that, with exception of the 5'-region, the FBP1-encoding locus in both cell lines is identical. The rearrangement in LL1 results from the insertion of an intracisternal A particle (IAP) in the head-to-head (antisense) orientation 72 base pairs (bp) upstream of the FBP1 ATG start codon. The IAP likely provides an alternative promoter for FBP1 expression which may produce a novel transcript with enhanced stability. Presence of the IAP appears to inactivate or relocate normal cis-acting regulatory sequences as expression of the FBP1 transcript in LL1 is not regulated by the folate status of the cell.

Amino Acid Sequence↗

PubMed1991

Defective endogenous retrovirus-like sequences and particles of Chinese hamster ovary cells.

The presence of budding C-type and intracytoplasmic A-type particles in Chinese hamster ovary (CHO) cells is well documented. However, extensive screening has failed to detect any evidence of infectivity. To investigate the origin and expression of these particles, retrovirus-like sequences which are actively transcribed in CHO cells have been cloned and characterized. Two families of sequences related to intracisternal A-particle (IAP) genomes of mice and Syrian hamsters were identified in cytoplasmic RNA from CHO cells (CHO IAP family I and family II). None of the four clones which were sequenced exhibited intact gag, pol, or env open reading frames. Only IAP family II sequences were present in purified extracellular particles of CHO cells. Several cDNA sequences related to mammalian C-type retrovirus genomes were isolated and cloned from gradient-purified, extracellular particles of recombinant CHO cells. All were homologous to the conserved endonuclease domain of murine leukemia virus. Nucleotide sequence analysis of the largest cDNA revealed multiple interruptions of the endonuclease encoding reading frame providing one possible explanation for the non-infectious nature of the particles observed in CHO cells. Both types of retrovirus-like sequences identified in purified extracellular particles of CHO cells (CHO IAP family II and C-type) were present as conserved, moderately repetitive sequences in DNA of all CHO cell lines examined, as well as in DNA from a Chinese hamster liver. It is therefore likely that the extracellular retrovirus-like particles of CHO cells are the products of endogenous provirus elements present in the germline of Chinese hamsters.

Animals↗

PubMed1979

Intracisternal A-particles in genetically diabetic mice: identification in pancreas and induction in cultured beta cells.

Sucrose density gradient analysis of purified pancreatic homogenates from glycaemic C57BL/Ks diabetes (db/db) mice and their normoglycaemic controls have revealed the presence in the diabetics of increased Mg++-dependent RNA-directed DNA polymerase activity sedimenting with a density of approximately 1.21 g/cm3. Electron microscopy revealed that this fraction contained typical intracisternal A-particles. Purified cultures of pancreatic islet cells 4--7 day old postnatal "misty diabetic" mice and normal siblings were established and then maintained in Eagle's minimal essential medium without serum. Under these conditions, the presence of intracisternal A-particles in beta cells of both mutant and control genotypes was very rare. No change in numbers of intracisternal A-particles was seen after 2--4 days of incubation in Dulbecco's-modified minimal essential medium containing 5.5 mmol/l glucose. However, when the glucose concentration of Dulbecco's medium was elevated to 16.5 mmol/l, ultrastructural changes specific to the beta cell population occurred that were reminiscent of those alterations observed in situ. Intracisternal A-particles were commonly seen in cultured beta cells showing hypersecretion-stress morphology. Since equal numbers of intracisternal A-particles were present in cultured beta cells from normal and mutant mice, it was concluded that the db gene itself was not required for intracisternal A-particle expression. The cell culture results suggest that elevated intracisternal A-particle activity observed in vivo may be produced directly or indirectly by the ambient high blood glucose levels characteristic of this mutant.

Animals↗

PubMed1991

Gene expression of an endogenous retrovirus-like element during murine development and aging.

We have measured intracisternal A-particle (IAP) RNA levels during development and aging in C57BL/6J mouse tissues to determine possible age-dependent changes in gene expression of these retrovirus-like sequences. Total RNA was isolated from tissues of embryonic and new born mice and mice ranging in age from 2 months to 32 months of age. RNA samples were either slot-blotted directly or fractionated on denaturing agarose gels and transferred to nylon membranes. Hybridization with cloned, 32P-labeled IAP sequences showed that both the mass amounts and the relative proportions of IAP transcripts varied between tissues and as a function of age. IAP gene products were higher in brain and kidney tissues than in liver and heart tissues. The relative proportion of transcripts increased in embryonic tissues until birth and following birth, was highest in neonatal or 2-month-old tissues. The adult levels of IAP-related RNAs did not change significantly from 6 to 24 months of age. However, 32-month-old tissues exhibited the lowest content of IAP transcripts, with the exception of heart tissue which did not change with age. A 5.4-kb RNA was the predominant IAP transcript in most samples, but each tissue had a characteristic size distribution of IAP-related transcripts. These results demonstrate that transcription of IAP genes continues throughout the life span of this mouse strain with tissue-specific and age-dependent regulation of expression.

Aging↗

PubMed1979

Tumorigenicity and intracisternal A-particle expression of hybrids between murine myeloma and lymphocytes.

Hybrids of BALB/c lymphocytes and a murine myeloma, a tumor that expresses intracisternal A-particles, were obtained with polyethylene glycol as the fusogen. The karyotype, tumorigenicity, and A-particle expression of the hybrid clones were assessed. All the hybrid clones analyzed were tumorigenic and expressed intracisternal A-particles even when they were the result of a fusion event between two lymphocytes and one myeloma cell in which no loss of chromosomes was detected. The tumors that developed following inoculation of hybrid cells into BALB/c mice (1 x 10(6) cells/mouse) were karyotypically identical to the inoculated cells. It appears that the two myeloma cell phenotypic traits analyzed (tumorigenicity and A-particle expression) are dominant.

Animals↗

PubMed2025

DNA methylation at retrotransposons protects the germline by preventing NRF1-mediated activation.

Silencing evolutionary young retrotransposons by cytosine DNA methylation is essential for spermatogenesis, as failure to methylate their promoters leads to reactivation, meiotic failure, and infertility. How retrotransposons reactivate in the absence of DNA methylation is poorly understood. We show that upon defective DNA methylation, distinct retrotransposon families display unique expression patterns and chromatin landscapes during mouse spermatogenesis. We find that their reactivation in meiotic spermatocytes correlates with the loss of bivalent H3K4me3-H3K27me3 chromatin marks. Through proteomics and chromatin profiling, we identify NRF1 as a DNA methylation-sensitive transcription factor that transactivates unmethylated retrotransposons. Conditional germline knockout of Nrf1 in the absence of DNA methylation rescues the silencing of the most mutagenic retrotransposon in mice, namely Intracisternal A-particle or IAP. Our findings reveal that chromatin modifications together with a DNA methylation-sensitive transcription factor regulate retrotransposon expression in the absence of DNA methylation in spermatogenesis, revealing a mechanism by which retrotransposons proliferate in the germline after evading DNA methylation-based silencing.

Animals↗