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PubMed1995

Expression of X-linked genes in androgenetic, gynogenetic, and normal mouse preimplantation embryos.

A quantitative RT-PCR approach has been used to examine the expression of a number of X-linked genes during preimplantation development of normal mouse embryos and in androgenetic and gynogenetic mouse embryos. The data reveal moderately reduced expression of the Prps1, Hprt, and Pdha1 mRNAs in androgenetic eight-cell and morula stage embryos, but not in androgenetic blastocysts. Pgk1 mRNA abundance was severely reduced in androgenones at the eight-cell and morula stages and remained reduced, but to a lesser degree, in androgenetic blastocysts. These data indicate that paternally inherited X chromosomes are at least partially repressed in androgenones, as they are in normal XX embryos, and that the degree of this repression is chromosome position-dependent or gene-dependent. Gynogenetic embryos expressed elevated amounts of some mRNAs at the morula and blastocyst stages, indicative of a delay in dosage compensation that may be chromosome position-dependent. The Xist RNA was expressed at a greater abundance in androgenones than in gynogenones at the eight-cell and morula stages, consistent with previous studies. Xist expression was observed in both androgenones and gynogenones at the blastocyst stage. We conclude that the developmental arrest in early androgenones may be, in part, due to reduced expression of essential X-linked genes, particularly those near the X inactivation center, whereas the developmental defects of gynogenones and parthenogenones, by contrast, may be partially due to overexpression of X-linked genes in extraembryonic tissues, possibly those farthest away from the X inactivation center.

Animals↗
PubMed2001

An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta.

A GFP transgene has been integrated on the proximal part of the mouse X chromosome just distal of Timp and Syn1. During development, this X-linked GFP transgene exhibits widespread green fluorescence throughout the embryonic and adult life of male mice but displays mosaic expression in tissues as a result of X-inactivation in females. In living female embryos, inactivation of the transgene is imprinted in extraembryonic regions and random in the embryo proper, demonstrating that this reporter is behaving in a similar fashion to the majority of X-linked loci, and so provides a vital readout of X chromosome activity. This is observation is further supported in T16H/X female mice harboring the GFP transgene on the normal X chromosome where reporter inactivation is observed in somatic cells. The differential expression of GFP activity facilitates fluorescence activated cell sorting for the purification of GFP+ vs. GFP- cells from female embryonic tissues, thereby allowing access to populations of cells that have kept active a particular X chromosome. By tracking the activity of this X-linked GFP transgene, we discovered that the primary and secondary giant cells of the X/X placenta maintain an active paternal copy of this transgene on the presumed silenced paternal X-chromosome. This finding implies that the imprint on the paternal X chromosome may be relaxed in these trophectodermal derivatives.

Animals↗
PubMed2001

Imprinting defects in mouse embryos: stochastic errors or polymorphic phenotype?

Defects in expression of imprinted genes are believed to cause developmental abnormalities and play a role in carcinogenesis. To determine whether spontaneous imprinting defects may occur in mouse embryos, we studied the expression of two imprinted genes H19 and Igf2 in individual postimplantation 7.5 d.p.c. and 8.5 d.p.c. embryos. Biallelic expression of H19 was found in 1.6% of the embryos, whereas biallelic expression of Igf2 was found in 0.5% of the embryos. The loss of H19 imprinting (LOI) observed in a small fraction of early postimplantation embryos may be purely stochastic. Alternatively, since we never observed it in an inbred background, it may depend on genetic factors acting in trans. Either mechanism could explain the occurrence of polymorphic imprinting as well as the genesis of sporadic imprinting defects, including cancer. The frequency of LOI of H19 was higher than the incidence of sporadic imprinting disorders in humans (about 1 in 20,000). This contradiction may be explained by different incidence of imprinting errors in different imprinted regions of the genome, in different species, or by loss of the majority of nonmosaic embryos with imprinting defects before birth.

Alleles↗
PubMed2001

Loss of imprinting and loss of heterozygosity on 11p15.5 in head and neck squamous cell carcinomas.

BACKGROUND: IGF2 and H19 are reciprocal imprinted genes with paternal and maternal monoallelic expression, respectively. This is interesting, because IGF2 is known as a growth factor, and H19 encodes a RNA with putative tumor suppressor action. Furthermore, IGF2 and H19 are linked genes located on chromosome 11p15.5, a common site of loss of heterozygosity in human cancers. METHODS: We performed an allelic-typing assay using a PCR-RFLP-based method for identification of heterozygous informative cases in head and neck squamous cell carcinomas. Tumoral total RNA was extracted from each of the heterozygotes and further studied by RT-PCR analysis. RESULTS: We detected the expression of the IGF2 gene in 10 of 10 informative cases. Two cases exhibited LOI of the IGF2 gene as evidenced by biallelic expression, and in another case, LOH was coupled with monoallelic expression of this growth factor. LOI for the H19 gene was observed in 1 of 14 informative samples analyzed. In this case, we also detected parallel monoallelic expression of the IGF2 gene. Down-regulation of the H19 gene was observed in 10 of 14 cases. CONCLUSION: These findings support the hypothesis that H19 may be a tumor suppressor gene involved in head and neck carcinogenesis. Furthermore, our data showed that genetic and epigenetic changes at 11p15.5 could lead to abnormal expression of imprinted genes in HNSCC.

Aged↗
PubMed1994

enod40, a gene expressed during nodule organogenesis, codes for a non-translatable RNA involved in plant growth.

Rhizobium meliloti can interact symbiotically with Medicago plants, thereby inducing root nodules. However, certain Medicago plants can form nodules spontaneously, in the absence of rhizobia. A differential screening was performed using spontaneous nodule versus root cDNAs from Medicago sativa ssp. varia. Transcripts of a differentially expressed clone, Msenod40, were detected in all differentiating cells of nodule primordia and spontaneous nodules, but were absent in fully differentiated cells. Msenod40 showed homology to a soybean early nodulin gene, Gmenod40, although no significant open reading frame (ORF) or coding capacity was found in the Medicago sequence. Furthermore, in the sequences of cDNAs and a genomic clone (Mtenod40) isolated from Medicago truncatula, a species containing a unique copy of this gene, no ORFs were found either. In vitro translation of purified Mtenod40 transcripts did not reveal any protein product. Evaluation of the RNA secondary structure indicated that both msenod40 and Gmenod40 transcripts showed a high degree of stability, a property shared with known non-coding RNAs. The Mtenod40 RNA was localized in the cytoplasm of cells in the nodule primordium. Infection with Agrobacterium tumefaciens strains bearing antisense constructs of Mtenod40 arrested callus growth of Medicago explants, while overexpressing Mtenod40 embryos developed into teratomas. These data suggest that the enod40 genes might have a role in plant development, acting as 'riboregulators', a novel class of untranslated RNAs associated with growth control and differentiation.

Amino Acid Sequence↗
PubMed2001

Frequent loss of imprinting of IGF2 and MEST in lung adenocarcinoma.

Genomic imprinting is a parental origin-specific chromosomal modification that causes differential expression of maternal and paternal alleles of a gene. Accumulating evidence suggests that deregulation of imprinted genes, including loss of imprinting (LOI), plays a role in oncogenesis. In the present study, we investigated allelic expression of six imprinted genes in human lung adenocarcinomas as well as in matched normal lung tissue. Informative cases showing heterozygosity for the gene of interest were selected from 35 patients. LOI of the insulin-like growth factor 2 gene (IGF2) and mesoderm-specific transcript (MEST, also known as paternally expressed gene 1) was noted in 47% (seven of 15) and 85% (11 of 13) of informative cases, respectively. Monoallelic expression was maintained in all the matched normal tissues examined. LOI of IGF2 was seen more frequently in moderately to poorly differentiated adenocarcinomas. In contrast, H19, small nuclear ribonucleoprotein-associated polypeptide N gene (SNRPN), necdin gene (NDN), and long QT intronic transcript 1 (LIT1) exhibited consistent monoallelic expression in all the informative samples. These findings indicated that independent deregulation took place in imprinted genes and suggested that aberrant imprinting of IGF2 and MEST was involved in the development of lung adenocarcinoma.

Adenocarcinoma↗
PubMed2001

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells.

3T3 Cells↗
PubMed1994

Expression of the Xist gene in urogenital ridges of midgestation male embryos.

We show by RT-PCR analysis that transcripts from the Xist gene, which is normally expressed from the inactive X chromosome of female somatic cells and postnatal male germ cells, are transiently expressed also in male embryos around the time of testis differentiation, mainly in somatic cells of urogenital ridges. In the postnatal testis, we find that Xist transcripts are mainly localized within the nucleus of haploid spermatids. These findings suggest that inactivation of the X-chromosome might occur not only in adult male germ cells, but also, transiently, in somatic cells of the male urogenital ridge. Both in the embryonal gonad and in differentiating germ cells Xist expression in males overlaps the pattern of expression of the testis determining gene Sry.

Animals↗
PubMed1996

Polymorphic functional imprinting of the human IGF2 gene among individuals, in blood cells, is associated with H19 expression.

In most non-neoplastic tissues studied to date, IGF2 is expressed only from the paternal allele and H19 is expressed only from the maternal allele. The choroid plexus, the only normal tissue to date where IGF2 is expressed from both parental alleles, does not express H19. We present an additional situation in which biallelic IGF2 expression is associated with the absence of H19 transcription in normal tissue: blood cells. In blood cells, functional IGF2 imprinting was found to be a polymorphic trait among individuals: expression was biallelic in 79 out of 85 individuals, but the remaining 6 expressed a single allele. Only the latter expressed H19. Finally, the familial clustering of functional IGF2 imprinting in blood cells suggests that the trait may be genotype-dependent.

Alleles↗
PubMed1997

A methylation-dependent DNA-binding activity recognising the methylated promoter region of the mouse Xist gene.

Differential methylation of CpG sites in the promoter region of the mouse Xist gene is correlated with Xist expression and X-chromosome inactivation in the female. Using oligonucleotides encompassing the differentially methylated sites as probes in band-shift assays, we have identified a nuclear protein which binds to a specific region of the promoter (between base pairs -45 and -30 upstream from the transcription start site) only when CpG sites within the CG rich region (GCGCCGCGG, -44 to -36) are methylated. Competition experiments with methylated or unmethylated heterologous oligonucleotides demonstrate that the activity is sequence-specific as well as methylation-dependent. Analysis by Southwestern blot identifies a protein of approximately 100 kDa molecular weight and confirms strong binding to the methylated Xist promoter oligonucleotide. Using a 233bp Xist-promoter luciferase construct in which the cytosines in the three CpG sites in the -44 to -36 region are mutated to thymine, we have established that this region is required for transcription from the mouse Xist promoter. Therefore, we suggest that the binding of the 100kDa protein to the methylated sequence leads to repression of transcription from the methylated Xist allele, thus suggesting a role in the regulation of both imprinted and random Xist transcription and X-chromosome inactivation.

Animals↗
PubMed1997

Mutational analysis reveals that an array of GCAAG/CTTGC motifs between sprit promoter sequences for RNA polymerase III is essential for neural BC1 RNA transcription.

BC1 RNA is expressed from an identifier (ID) sequence by RNA polymerase III (Pol III) and occurs in neural cells as a ribonucleoprotein particle (BC1 RNP). On the BC1 RNA gene, between the Pol III promoter A and B boxes, there is a region which contains short inverted repeats, including three GCAAG/CTTGC motifs. We found that a nuclear protein binds specifically to this region and, using an in vitro transcription system, demonstrated that point mutations within these motifs markedly inhibit BC1 RNA transcription. These results suggest that the GCAAG/CTTGC motif region and its binding protein may play a role in the transcription of BC1 RNA. Moreover, we demonstrated that transcription is repressed by a concomitant molar excess of BC1 RNA and that the BC1 RNA transcribed by this system forms an RNP with nuclear protein(s), suggesting some interaction of BC1 RNA with transcription factor(s).

Animals↗
PubMed1998

Difference in chromatin packaging between active and inactive X chromosomes by fractionation and allele-specific detection.

Using a novel method consisting of chromatin fractionation and allele-specific detection, chromatin packaging is compared between active X (Xa) and inactive X (Xi) chromosomes for five tumor cell clones that were derived from inter-subspecific F1 female mice. Separation of heterochromatic (H) and euchromatic (E) fractions is monitored by hybridization with subtelomeric satellite DNA and ribosomal RNA gene and by PCR amplification of p53 gene/pseudogene with one primer set. The H fraction was enriched with satellite and p53 pseudogene probably existing in heterochromatic regions while the E fraction showed inverse, suggesting fair separation. Analysis with seven marker and three gene loci revealed concentration of alleles on Xi in the H fraction and those on Xa in the E fraction, though the concentration levels varied. This implies that the packaging level of Xi is higher than that of active or inactive euchromatin on Xa. Intriguingly, one cell line showed biallelic expression and chromatin relaxation of the Pgk-1 locus, suggesting that the relaxation occur regionally on X chromosome.

Alleles↗
PubMed1998

An E-box sequence acts as a transcriptional activator for BC1 RNA expression by RNA polymerase III in the brain.

BC1 RNA is a small cytoplasmic RNA that is transcribed by RNA polymerase III (Pol III) in the rodent nervous system. In addition to essential intragenic promoter elements for Pol III, the BC1 RNA gene has five E-box sequences (CANNTG) in its 5' flanking region. Deletion analysis using an in vitro transcription system revealed that the region containing the E2 site (CAATTG) was necessary for effective transcription of BC1 RNA. A construct with point mutations within the E2 site showed reduced transcriptional activity. Furthermore, DNaseT I protection and gel retardation assays demonstrated that the E2 site was recognized specifically by a brain nuclear protein(s). These results suggest that the upstream E-box sequence and its binding protein may be involved in the regulation by Pol III of preferential BC1 RNA expression in the brain.

Animals↗
PubMed1998

BC1 RNA protein particles in mouse brain contain two y-,h-element-binding proteins, translin and a 37 kDa protein.

Neural BC1 RNA is distributed in dendrites in the form of ribonucleoprotein particles (RNP). Recently, Han et al. reported that testis-brain RNA-binding protein (the mouse homologue of Translin), known to be a translational repressor, links a subset of mRNAs to microtubules (MTs) through binding to their y-,h-sequence elements. We found that similar elements are also present in BC1 RNA. Therefore, it is possible that they also act as cis-elements, together with their transacting proteins, and control the transport of BC1 RNA along dendritic MTs. In this study, we demonstrated that two y-,h-element-binding proteins copurified with BC1 RNP. One was identified as mouse Translin and the other was a 37 kDa protein. This macromolecular assembly may constitute a transport particle for BC1 RNA. It is also possible that BC1 RNP or Translin itself may play a regulatory role(s) in the translation of mRNAs within dendrites.

Animals↗
PubMed1998

The dendritic translocation of translin protein in the form of BC1 RNA protein particles in developing rat hippocampal neurons in primary culture.

Neural BC1 RNA is distributed in neuronal dendrites as ribonucleoprotein particles (RNP). Our previous studies indicated the presence of Translin in BC1 RNPs, which is a translational repressor and links a subset of mRNAs to microtubules. In this study, we confirmed that Translin associates with BC1 RNP and we used immunocytochemical methods to examine the subcellular distribution of Translin in developing hippocampal cells in primary cultures. Translin was detected in both the nuclei and cytoplasm of neurons, whereas in glial cells it was localized in the nuclei. Consistent with the reported developmental time course of BC1 RNA expression and dendritic delivery the translocation of Translin to the neuronal dendrites appeared to correlate with neuronal development and differentiation events such as the onset of synaptogenesis in culture. These observations suggest that BC1 RNP or Translin itself may be relevant to the dendritic translation of mRNAs in response to transsynaptic activity.

Animals↗
PubMed1999

Proinsulin-like growth factor-II overexpression does not alter monoallelic H19 gene expression in transfected human embryonic kidney fibroblasts.

Insulin-like growth factor-II (IGF-II) is a potent mitogen for cells in culture. The H19 gene is a developmentally regulated gene with putative tumor suppressor activity, and loss of H19 expression may be involved in tumorigenesis. The H19 gene is closely linked to the human IGF-II gene (IGF2) on chromosome 11p15.5 and these genes are reciprocally imprinted in most fetal tissues. H19 is expressed only from the maternal and IGF2 from the paternal chromosome. We have asked whether overexpression of proIGF-II alters H19 imprinting status and/or expression. Human embryonal kidney fibroblasts (293 cells) were stably transfected with a PCMV5 vector containing the full length human IGF-II cDNA or a control cDNA. Transfectant clones expressed large quantities of IGF-II mRNA and secrete 1-5 ug/ml and 150-230 ng/ml proIGF-II within 24 hours of serum-free culture (transfectant 293-9 and -11 respectively) (1). Cells were genotyped at the exon 5, RsaI restriction fragment length polymorphism (RFLP) and found to be informative (+/-). H19 expression was monoallelic (+) indicating preservation of H19 imprinting in all cell lines. Using quantitative RT-PCR with internal competitors for H19 and for IGF-II cDNA, overexpression of IGF2 in 293-11 and 293-9 cells was confirmed. In contrast, no significant difference with respect to H19 expression was detected between the overexpressing cells and control lines. In conclusion, (1) human embryonal fibroblasts express the H19 gene. (2) H19 imprinting is preserved in these cells. (3) proIGF-II overexpression does not alter H19 expression.

Alleles↗
PubMed1999

An antisense transcript to SMAD5 expressed in fetal and tumor tissues.

SMAD5, a transducer of TGF-beta/BMP inhibitory signals and a tumor suppressor candidate, localizes to the region of invariant loss in human myeloid neoplasms, on chromosome 5q31.1. Recent evidence indicates a gene-dosage effect along the TGF-beta/BMP signaling pathways. We have identified a novel transcript designated DAMS, whose 3' exonic sequences contain in part an alternate 5' exon of SMAD5, in the antisense orientation. Expressed sequenced tags (ESTs) for DAMS are found in fetal tissues (heart, adrenal glands, and total fetus) and pancreatic tumor cDNA libraries. In contrast to SMAD5, DAMS expression is not readily detectable in adult and fetal tissues. Semiquantitative PCR suggests that the stoichiometry between SMAD5 and DAMS transcripts ranges between 15 and 120 in normal and malignant hematopoietic cells. The findings raise the possibility that DAMS may be a fail-safe mechanism for precise regulation of SMAD5 transcript levels that may be critical in maintaining normal homeostasis.

5' Untranslated Regions↗