A spontaneous mutation causing deafness and circling behavior was discovered in a C3H/HeJ colony of mice at the Jackson Laboratory. Pathological analysis of mutant mice revealed gross morphological abnormalities of the inner ear, and also dysmorphic or missing kidneys. The deafness and abnormal behavior were shown to be inherited as an autosomal recessive trait and mapped to mouse chromosome 1 near the position of the Eya1 gene. The human homolog of this gene, EYA1, has been shown to underly branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by hearing loss with associated branchial and renal anomalies. Molecular analysis of the Eya1 gene in mutant mice revealed the insertion of an intracisternal A particle (IAP) element in intron 7. The presence of the IAP insertion was associated with reduced expression of the normal Eya1 message and formation of additional aberrant transcripts. The hypomorphic nature of the mutation may explain its recessive inheritance, if protein levels in homozygotes, but not heterozygotes, are below a critical threshold needed for normal developmental function. The new mouse mutation is designated Eya1(bor) to denote its similarity to human BOR syndrome, and will provide a valuable model for studying mutant gene expression and etiology. (+info)
(2/124) Intracisternal type A particle-mediated activation of the Notch4/int3 gene in a mouse mammary tumor: generation of truncated Notch4/int3 mRNAs by retroviral splicing events.
The int3 oncogene was discovered as a frequent target in mouse mammary tumor virus-induced mammary tumors and encodes the intracellular domain of a Notch4/int3 protein. In one spontaneous mammary tumor, no. 9, that developed in a BALB/c mouse, we have found an insertion of a 1.2-kb sequence, consisting of a 5' long terminal repeat and gag sequences of an intracisternal type A particle (IAP) as well as an extra copy of the Notch4/int3 genomic sequences containing exons 23 and 24, into the intron between exons 24 and 25 of the Notch4/int3 gene. In this tumor, unique splicing events between the IAP and the Notch4/int3 sequences generated two types of IAP-Notch4/int3 fusion transcripts encoding two different portions of the intracellular domain of Notch4/int3 proteins: one with a RAM domain and the other without. Interestingly, these two proteins showed different subcellular localizations in a mouse mammary epithelial cell line, HC-11. (+info)
(3/124) Characterization of a mammalian gene related to the yeast CCR4 general transcription factor and revealed by transposon insertion.
Murine intracisternal A-particles (IAPs) are reiterated retrovirus-like transposable elements that can act as insertional mutagens. Accordingly, we previously identified a chimeric transcript initiated at an IAP promoter and extending through a 3'-located open reading frame with significant similarity to the C-terminal domain of the yeast CCR4 general transcription factor. In this report, we characterize the corresponding murine gene, mCCR4, and its human homologue, thus providing the first description of CCR4-like factors in mammals. cDNA cloning revealed two mCCR4 mRNAs of 2.7 and 3.1 kilobases, differing by their transcription start sites within the native mCCR4 gene promoter, and encoding a putative 430-amino acid protein. The mCCR4 gene contains three exons and two introns spanning almost 27 kilobases. The IAP insertion, detected only in some laboratory mouse strains, is recent and lies within the first intron. The 5'-region of the gene has features of housekeeping gene promoters. It lacks a TATA box but contains a CpG island and Sp1 sites. This region discloses strong promoter activity in transient transfection assays and also stimulates transcription in the reverse orientation, a feature common to other CpG island-containing promoters. Transcripts were detected in all the organs tested, although at a variable level, and displayed no strain-dependent differences relative to the IAP insertion, suggesting the existence of mechanisms preserving mCCR4 transcription from the usually deleterious effects of intronic transposition. The strong amino acid conservation between the human, murine, and the previously identified Xenopus CCR4-like proteins, is consistent with an important and conserved role for this protein in vertebrates. (+info)
(4/124) Oligonucleotide scanning of native mRNAs in extracts predicts intracellular ribozyme efficiency: ribozyme-mediated reduction of the murine DNA methyltransferase.
Modulation of gene expression by catalytic RNA requires accessible ribozyme cleavage sites in the target mRNA, and accessibility is determined by the secondary and tertiary structure of the target RNA, as affected by its interactions with cellular proteins. As we previously reported, an oligonucleotide-scanning approach using antisense oligonucleotides can be used to determine RNA accessibility in cell extracts. To test whether this method can be used to improve selection of ribozyme target sites, we designed ribozymes corresponding to the sites identified by oligonucleotide scanning and have evaluated their catalytic activities, first in cell extracts and then in transduced cell lines. As a target we used the mRNA of murine DNA (cytosine-5)-methyltransferase 1 (MTase). For intracellular studies, the ribozyme genes were inserted downstream of a Pol III tRNAVAL promoter, which in turn was cloned in the U3 region of a retroviral vector. We find that the efficiency of the ribozymes both in cell extracts and in vivo corresponds with the relative effectiveness predicted by the oligonucleotide-scanning assay. The best ribozyme causes a 70-80% reduction in the MTase mRNA levels in NIH 3T3 cells that are stably transduced with the retroviral constructs. This reduction in mRNA levels is accompanied by a small decrease in the methylation of repetitive intercisternal A particle DNA elements. Ribozyme expression also increased several-fold the reactivation frequency of a methylation-silenced green fluorescent protein (GFP) transgene. Both the reduction in methylation and reactivation of GFP were roughly equivalent to the effects obtained by treating NIH 3T3 cells with 2.5 microM 5-azacytidine, which gives an effect of about 10% of maximum. These results confirm the validity of the cell extract approach for ribozyme site selection and provide a potentially useful ribozyme for future study of DNA methyltransferase function. (+info)
(5/124) Differential expression of intracisternal A-particle transcripts in immunogenic versus tumorigenic S49 murine lymphoma cells.
Tumorigenic S49 mouse lymphoma cells (T-25) were compared to their nontumorigenic (immunogenic) substrate-adherent descendants (T-25-Adh), using the differential display technique. A 784-bp fragment with 92% sequence homology to the intracisternal A-particle (IAP) element family was isolated from the latter cells. IAP sequences are endogenous, noninfectious retroviral elements that can undergo transpositions and act as mutagens. Expression of IAP transcripts (as detected by the isolated fragment) was 5- to 10-fold higher in T-25-Adh cells than in T-25 cells. IAP RT-PCR cDNA clones derived from the immunogenic T-25-Adh cells, but not from T-25 cells, contain two distinctive motifs: (i) a motif characteristic of IAP elements expressed in lymphoid cells (lymphocyte specific, LS); (ii) a nonapeptide sequence known to stimulate cytotoxic T lymphocytes in a leukemia cell line expressing IAP sequences. In addition, expression of transcripts containing these motifs is enhanced in the immunogenic cells as opposed to the tumorigenic cells. Furthermore, one of the IAP elements (belonging to the LS1 subfamily) is specifically hypomethylated in the DNA of the immunogenic cells. The above-mentioned relationship was strengthened when tumorigenic revertants derived from T-25-Adh cells, as well as independently selected tumorigenic and immunogenic S49 sublines, were studied. In all cases, enhanced immunogenicity was linked to the up-regulation of specific IAP elements. No transpositions of LS1 elements were observed among the different sublines studied. These findings suggest that, in the S49 lymphoma, selectively expressed IAP retroviral elements may function in a tumor suppressive capacity by affecting the immunogenic potential of these cells. (+info)
(6/124) Defective spectrin integrity and neonatal thrombosis in the first mouse model for severe hereditary elliptocytosis.
Mutations affecting the conversion of spectrin dimers to tetramers result in hereditary elliptocytosis (HE), whereas a deficiency of human erythroid alpha- or beta-spectrin results in hereditary spherocytosis (HS). All spontaneous mutant mice with cytoskeletal deficiencies of spectrin reported to date have HS. Here, the first spontaneous mouse mutant, sph(Dem)/ sph(Dem), with severe HE is described. The sph(Dem) mutation is the insertion of an intracisternal A particle element in intron 10 of the erythroid alpha-spectrin gene. This causes exon skipping, the in-frame deletion of 46 amino acids from repeat 5 of alpha-spectrin and alters spectrin dimer/tetramer stability and osmotic fragility. The disease is more severe in sph(Dem)/sph(Dem) neonates than in alpha-spectrin-deficient mice with HS. Thrombosis and infarction are not, as in the HS mice, limited to adults but occur soon after birth. Genetic background differences that exist between HE and HS mice are suspect, along with red blood cell morphology differences, as modifiers of thrombosis timing. sph(Dem)/sph(Dem) mice provide a unique model for analyzing spectrin dimer- to-tetramer conversion and identifying factors that influence thrombosis. (+info)
(7/124) Enhanced expression of the early retrotransposon in C3H mouse-derived myeloid leukemia cells.
Cells of acute myeloid leukemia (AML) from C3H/He mice express an increased amount of RNA for an endogenous retrovirus-like retrotransposon, intracisternal A-particle element. We analyzed the transcription of other mouse retrotransposons in C3H-derived tumor cells and found that all the AML lines from different mice overexpress early-transposon (ETn) RNA. In contrast, only faint levels of ETn were detected in the cells from other tumors, including hepatoma and lymphoma. The polyadenylation sites of the ETn RNA in the AML cells varied. We also determined the binding site for the nuclear extract of the AML cells in the long terminal repeat sequence of ETn. The overexpression of ETn as a common phenotype of AML cells suggests that myeloid cells with this phenotype are the origin of all the AML cells or that the phenotype is acquired during leukemogenesis. (+info)
(8/124) Identification of a novel posttranscriptional regulatory element by using a rev- and RRE-mutated human immunodeficiency virus type 1 DNA proviral clone as a molecular trap.
Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a rev- and RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote replication of the Rev- and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export from Xenopus oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways, these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional control element that uses a conserved cellular export mechanism. (+info)