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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↗
PubMedDate not supplied

Paradoxical effects of recombinant mouse beta-interferon on different expression levels of intracisternal A particle genes.

We have studied the effect of mouse recombinant beta-Interferon on the expression of the intracisternal A particle (IAP) genes in a transformed mouse fibroblast cell line. Northern and immunoprecipitation analysis showed an increase in specific transcripts and polypeptides. Run-on experiments on isolated nuclei and transfection assays with an IAP long terminal repeat construct, coupled with the chloramphenicol acetyl transferase gene, showed no enhancement of the transcription rate following beta-IFN treatment. This suggests that the increase in IAP expression depends on a post-transcriptional event. Electron microscopic analysis revealed that in spite of the higher levels of IAP related polypeptides, the number of particles was significantly reduced by interferon.

Animals↗
PubMed1990

The activated form of p53 is not a transactivator of the intracisternal A particle long terminal repeat promoter.

Published observations have suggested that the activated form of p53 protein could transactivate the Intracisternal A particle long terminal repeat promoter (IAP-LTR). In this paper we demonstrate that the increased expression from this promoter was due to effects of the co-transferred plasmids and not p53 per se. In transient expression experiments, co-transfer of either a p53 or a p53 frame-shift mutant plasmid with an IAP-LTR driven chloramphenicol acetyl transferase (CAT) plasmid gave a similar increase in CAT activity. Further, this increase in CAT gene activity could also be achieved by co-transfer of a plasmid containing a viral promoter alone.

Chloramphenicol O-Acetyltransferase↗
PubMed1990

Retrotransposons as mutagens in the induction of growth autonomy in hematopoietic cells.

Factor-independent mutants of hematopoietic cells, especially of multipotent cells, are valuable tools to identify genes that regulate stem cell proliferation and differentiation and thus may be important in leukemogenesis. Factor-independent mutants from both myeloid precursor and hematopoietic stem cell lines were isolated. The frequency of such mutants in a given cell population was one to two orders of magnitude lower for the multipotent cell line FDC-Pmix (3.6 x 10(-9)) than for the myeloid precursors, FDC-P1-M (1.7 x 10(-8)) and D35 (2.2 x 10(-7)). Analysis of these mutants revealed several mechanisms by which growth autonomy was obtained, either with or without direct contribution of growth factor gene activation. The molecular basis of spontaneous activation of the Multi-CSF (Interleukin3) gene was determined and compared to activation of the GM-CSF gene in a previous study. Multi-CSF gene activation in both precursor and stem cells was caused by the insertion of an intracisternal A particle (IAP) provirus. In two independent mutants of the D35 cell line, activation of the Multi-CSF or the GM-CSF gene was caused by almost identical IAPs with a 99% homology in the U3 and R region of the long terminal repeat. This result demonstrates that only one class of IAPs, or perhaps a single provirus, is involved in transposition and gene activation in a particular cell line. A unique example of anti-sense promotion from an IAP provirus in one Multi-CSF mutant underlines the versatility of these elements as natural insertional mutagens.

Amino Acid Sequence↗
PubMed1985

AtT20 pituitary tumour cells contain mouse mammary tumour virus and intracisternal A-type particles in addition to murine leukemia virus.

By electron microscopy and immunocytochemistry we have examined the retroviruses endogenous to AtT20 D16V cells, a cloned line of murine pituitary tumour cells. In addition to the C-type retrovirus particles related to Rauscher murine leukemia virus (MuLV) previously reported to bud from these cells we observed cytoplasmic A-type particles and intracisternal A-type particles (IAP). In the cytoplasm the A-type particles occur in large clusters often associated with sheets of material with a fine structure resembling the shells of the particles. At the plasma membrane individual A-type particles bud to give rise to extracellular virions. The IAP are restricted to the rough endoplasmic reticulum (RER) into which they bud: they are not transported out of the RER to the Golgi apparatus and beyond. We describe a new monoclonal antibody (designated 83E7) which is specific for an epitope of the major core protein (MTVp27) of mouse mammary tumour virus (MMTV). Using immunogold labelling procedures we have specifically labelled both the A-type particles and the associated sheets of material with this antibody. We conclude that the A-type particles and the virions they give rise to are MMTV. The sheets of material must also at least in part be made up of the major core protein of MMTV or its precursor polypeptide. AtT20 cells, therefore, contain endogenous MuLV and MMTV as well as IAP.

Animals↗
PubMed1986

[Nature of the endogenous retrovirus-like particles of the rat liver].

Previously virus-like particles (VLP) with properties resembling retroviruses were isolated from the liver of Wistar rats. Molecular hybridization and CRIA test were used for further analysis of the VLP. The CRIA method showed that VLP preparation lacked antigenic determinants of the major internal protein of C-type virus. By the dot hybridization technique no homology was detected between VLP and Mo-MuLV DNA, however VLP RNA was found to be homologous to IAP (interstitial A particles) DNA of mice. VLP proviral DNA was detected in the rat genome by the blot hybridization technique. Thus, it was concluded that VLP resemble IAP. A possible role of rat IAP expression is discussed.

Animals↗
PubMed1986

Activation of oncogenes by transposable elements.

Mammalian DNA contains several families of highly repeated sequences, some of which have been suggested to be mobile elements. We have screened tumour tissue for the rearrangement of cellular oncogenes and found evidence for the behaviour of repetitive DNA sequences as transposable elements which may activate oncogenes. In the mouse myeloma NSI and XRPC24 we found that intracisternal A particle genome was inserted into the coding region of c-mos. In both cases the rearranged c-mos was transcriptionally activated and was also able to transform NIH 3T3 cells. In the canine transmissible venereal tumour we found that c-myc was rearranged due to the insertion of an 1.8 kilobase pair cellular DNA. Nucleotide sequence analysis demonstrated that the inserted piece is 60% homologous to the monkey KpnI element which is a representative of the LINE group.

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↗
PubMed1990

Establishment of an interleukin-5-dependent subclone from an interleukin-3-dependent murine hemopoietic progenitor cell line, LyD9, and its malignant transformation by autocrine secretion of interleukin-5.

An interleukin-5 (IL-5)-dependent subclone, K-5, was established from an IL-3-dependent murine hemopoietic progenitor cell line by co-culturing with bone marrow stroma cells. K-5 cells were induced to differentiate into myeloid lineage cells by co-culturing with cloned PA6 stroma cells. By co-culturing with another cloned stroma cell (ST-2s10), K-5 cells gave rise to a factor-independent transformant cell line LT-5 which proliferated in an autocrine manner by secretion of IL-5 and produced tumors in nude mice. Molecular cloning of the IL-5 gene of LT-5 cells and the nucleotide sequencing of its 5' flanking region indicate that a transposition of an intracisternal A-particle (IAP) element to the 5' flanking region of the IL-5 gene is responsible for the constitutive expression of IL-5 mRNA of an aberrant size in LT-5 cells.

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↗
PubMed1990

Submegabase clusters of unstable tandem repeats unique to the Tla region of mouse t haplotypes.

We describe here the identification and genomic organization of mouse t haplotype-specific elements (TSEs) 7.8 and 5.8 kb in length. The TSEs exist as submegabase-long clusters of tandem repeats localized in the Tla region of the major histocompatibility complex of all t haplotype chromosomes examined. In contrast, no such clusters were detected among 12 inbred strains of Mus musculus and other Mus species; thus, clusters of TSEs represent the first absolutely qualitative difference between t haplotypes and wild-type chromosomes. Pulsed field gel electrophoresis shows that the number of clusters, and the number of repeats in each cluster are extremely variable. Dramatic quantitative differences of TSEs uniquely distinguish every independent t haplotype from any other. The complete nucleotide sequence of one 7.8-kb TSE reveals significant homology to the ETn (a major transcript in the early embryo of the mouse), and some homologies to intracisternal A-particles and the mammary tumor virus env gene. Apart from the diagnostic relevance to t haplotypes, evolutionary and functional significances are discussed with respect to chromosome structure and genetic recombination.

Animals↗
PubMed1990

Isolation of genes highly expressed in early and late stages of Friend virus-induced erythroleukemia in mice.

We isolated two genes that are highly expressed in Friend erythroleukemia. The first one, expressed at an extremely high level in a late-stage tumorigenic cell line F5-5, was found to be a species of intracisternal A-particle. Most of those genomes in F5-5 DNA were not amplified nor rearranged compared with those in the normal cell DNA, suggesting transcriptional activation. Another late-stage cell line T3K showed similar results. The second gene, highly expressed in early-stage cells but weaker in late-stage cells, was identified as one of the heme-synthesizing enzymes, uroporphyrinogen decarboxylase. Usefulness of these two genes for the analysis of erythroid transformation and differentiation will be discussed.

Animals↗
PubMed1987

Nearly identical members of the heterogeneous IAP gene family are expressed in thymus of different mouse strains.

Poly(A)RNAs prepared from the thymuses of C57BL/6J and DBA/2J mice were used to construct cDNA libraries in the bacterial expression vector lambda gt11. The libraries were scanned first for protein production with polyvalent antiserum prepared against the 73kDa gag protein of mouse intracisternal A-particles (IAP). Reactive plaques were crossed-screened by hybridization with an IAP-specific DNA probe. Two IAP-specific protein-producing plaques were obtained from the C57BL/6 library and 4 from the DBA/2 library. One C57BL/6 cDNA clone (B12) and two DBA cDNA clones (D8 and D20) were sequenced in their entirety. Clones B12 and D8 were remarkably similar, particularly when compared to the 6 other IAP elements that have been sequenced thus far. We discuss the evidence which leads us to suggest that these clones may be derived from allelic IAP elements expressed in mouse thymus.

Alleles↗
PubMed1986

Nucleotide sequence of human endogenous retrovirus genome related to the mouse mammary tumor virus genome.

We determined the complete nucleotide sequence of the human endogenous retrovirus genome HERV-K10 isolated as the sequence homologous to the Syrian hamster intracisternal A-particle (type A retrovirus) genome. HERV-K10 is 9,179 base pairs long with long terminal repeats of 968 base pairs at both ends; a sequence 290 base pairs long, however, was found to be deleted. It was concluded that a composite genome having the 290-base-pair fragment is the prototype HERV-K provirus gag (666 codons), protease (334 codons), pol (937 codons), and env (618 codons) genes. The size of the protease gene product of HERV-K is essentially the same as that of A- and D-type oncoviruses but nearly twice that of other retroviruses. A comparison of the deduced amino acid sequences encoded by the pol region showed HERV-K to be closely related to types A and D retroviruses and even more so to type B retrovirus. It was noted that the env gene product of HERV-K structurally resembles the mouse mammary tumor virus (type B retrovirus) env protein, and the possible expression of the HERV-K env gene in human breast cancer cells is discussed.

Amino Acid Sequence↗
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↗