DNA methylation contributes to tissue- and allele-specific expression of the T-cell differentiation marker RT6

We investigated the role of DNA methylation in gene regulation of the rat T-cell differentiation marker RT6. Analysis of the methylation status of various tissues revealed that the RT6 promoter was hypomethylated in RT6-expressing tissues, and methylated in nonexpressing ones. Remarkably, among RT6-nonexpressing tissues, the extent of methylated regions varied greatly between lymphatic tissues, where regions larger than 23 kb were methylated, and nonlymphatic tissues, where methylation was restricted to a 3- to 4-kb region surrounding the promoter. We have previously shown that cis-regulatory elements determine differential expression of the two RT6 alleles in a subpopulation of T cells. We now show that the RT6 alleles in these cells differed in their methylation status. The promoter region of the silent allele was methylated, while that of the transcribed allele was not. Upon treatment of RT6-nonexpressing thymoma cells with the methyltransferase inhibitor 5-azacytidine, RT6 expression was induced. In RT6 heterozygous hybridoma cells, expressing only one RT6 allele, induction of the silent, methylated RT6 allele was observed. Sensitivity of the RT6 promoter to DNA methylation was demonstrated by promoter-specific in vitro methylation, which inhibited RT6 promoter activity, while that of the SV40 promoter was not influenced. Our findings indicate that DNA methylation plays an important role in the control of monoallelic and tissue-specific RT6 expression.     

BglII identifies a frequent biallelic DNA polymorphism of the human RT6 gene

A genomic probe of the human RT6 gene detects a frequent biallelic BglII polymorphism. Allele A has a frequency of 63%, whereas that of allele B is 37%. This restriction fragment length polymorphism provides the first known genetic marker for this gene.     

Chromosomal localization, expression pattern, and promoter analysis of the mouse gene encoding adipocyte-specific secretory protein Acrp30

Acrp30 is an abundantly expressed secretory protein exclusively synthesized in adipose tissue. Due to the dysregulation in various forms of obesity in humans and mice and its strong structural similarity to TNFalpha, it is currently under study as an important molecule involved in whole body energy homeostasis. Here we describe the sequence of mouse Acrp30 locus, define the intron/exon boundaries and map the gene to the telomere of mouse chromosome 16, syntenic to the human chromosomal locus 3q27. We demonstrate that alternative polyadenylation gives rise to two distinct mRNA species. We also show that Acrp30 expression is induced only at the late stages of mouse embryonic development. Finally, we have characterized the mouse Acrp30 promoter in tissue culture cells. We propose that Acrp30 promoter has the potential to drive strong adipocyte specific heterologous gene expression in transgenic mice.     

Chromosomal assignment and regional localization of myeloperoxidase in the mouse

Using a rat x mouse somatic cell hybrid that contains chromosome 11 as the only mouse material, we have shown that myeloperoxidase, which maps to human chromosome 17, maps to mouse chromosome 11. Regional assignment of the gene by in situ hybridization localized Mpo to the region C-E1, with a peak at band 11C. These results further confirm and extend observations on the remarkable homology between human chromosome 17 and mouse 11.     

Chromosomal localization of the mouse gene coding for vimentin

The chromosomal location of the mouse gene coding for vimentin, one of the intermediate filament subunits, was determined by in situ hybridization using specific H3-labelled DNA probes. There is only one copy of the vimentin gene and it is located on chromosome 2 region A2.     

Chromosomal localization of mouse and human neurotensin receptor genes

Neurotensin is a tridecapeptide that plays several neurotransmitter or neuromodulatory roles both in the central nervous system and in the periphery. These actions are mediated by a high-affinity receptor (Ntsr). Both rat and human cDNAs encoding high-affinity receptors have been recently cloned. The availability of Ntsr probes allowed us to localize the corresponding genes on the mouse and human chromosomes. The present data demonstrate that the Ntsr gene is assigned to the H region of the mouse Chromosome (Chr) 2 and to the long arm of the human Chr 20.     

Characterization of multiple alleles of the T-cell differentiation marker ART2 (RT6) in inbred and wild rats

ART2 (RT6) belongs to the family of mono-ADP-ribosyltransferases (ARTs). ART2 is a T-cell differentiation marker expressed by the majority of mature peripheral T cells in the rat. The two known ART2 allotypes display approximately 95% amino acid identity. We sequenced the ART2 coding regions from 18 inbred rat strains and found two additional alleles, termed Art2 ^a2 and Art2 ^b2 . Monoclonal antibody Gy12/61 specifically reacted with Art2 ^a2 but not Art2 ^a1 lymph node cells. Expression of ART2 allotypes in Jurkat cells confirmed this specificity. A polymerase chain reaction (PCR) assay using restriction fragment length polymorphisms is described, which allows the easy discrimination of Art2 alleles. All four laboratory rat alleles, as well as an additional sequence variant, were found amongst 18 wild rat DNA samples. PCR analysis confirmed the selective presence of a rodent identifier (ID) element in the Art2 ^a but not the Art2 ^b alleles in all rats studied. Analysis of Art2 ^a1 and Art2 ^b2 genes showed greater divergence in coding than in non-coding regions. Together with the finding of a high number of non-synonymous mutations leading mostly to non-conservative amino acid substitutions clustered on the side facing away from the cell surface, this suggests that the Art2 polymorphism has been subject to selection.     

Chromosomal localization of opioid peptide and receptor genes in the mouse

Opiate receptors are the primary targets for the drugs of abuse morphine and heroin. In this study, we completed the localization on mouse chromosomes of the genes encoding mu (Oprm) and kappa (Oprk) receptors, as well as the genes for the opioid propeptides proenkephalin (Penk) and prodynorphin (Pdyn). The genetic mapping was performed using a panel of DNA samples from an interspecific cross [C3H/HeJ-gld and (C3H/HeJ-gld x Mus spretus)Fi] that has been characterized for more than 800 markers throughout the genome. The genes are localized on mouse Chr 1 (Oprk, 10 cM from the centromere), Chr 2 (Pdyn, 75 cM from the centromere), Chr 4 (Penk, 1 cM from the centromere) and Chr 10 (Oprm, 10 cM from the centromere). Interestingly, the gene for the mu receptor is located in the same region as a Quantitative Trait Locus for high morphine consumption, thus raising the possibility of its direct role in drug abuse mechanisms.     

Chromosomal distribution, localization and expression of the human endogenous retrovirus ERV9

ERV9 is a class I family of human endogenous retroviral sequences. Somatic cell hybrid genomic hybridization experiments using a mono-chromosomal panel indicate the presence of approximately 120 ERV9 loci in the human genome distributed on most chromosomes. Fluorescence in situ hybridization (FISH) using an ERV9 cDNA probe containing gag, pol and env sequences, verified this observation and a consistent signal was found at the chromosome region 11q13.3-->q13.5. By analysis of a panel of radiation hybrids, an ERV9 locus was mapped to within a 300-kbp region at the chromosome site 11q13. The marker cCLGW567 and the locus MAP3K11/D11S546 centromeric and telomeric flanked it, respectively. Northern blot analysis, using an ERV9 LTR probe, indicated that most normal tissues examined expressed low abundant ERV9 LTR driven mRNAs of various sizes. The most prominent expression was found in adrenal glands and testis. However, the level of expression varied in the same tissues among different individuals indicating that ERV9 mRNA expression probably is inducible in certain tissues or at various cell stages.     

Tonofilament protein from newborn rat epidermis. Isolation, localization, and biosynthesis of marker of epidermal differentiation.

A novel extraction procedure, previously used on the cell walls of dermatophytes, has been applied to the epidermis of newborn rat. A leucine-rich fraction was isolated which contained over 60% of the total epidermal radioactivity from [3H]leucine in 15 to 20% of the total protein. This fraction was further purified by chromatography in DEAE-cellulose and Sephadex G-200. The protein with the highest specific activity from [3H]leucine was isolated and gave a single band in sodium dodecyl sulfate polyacrylamide gels of molecular weight = 58,000. Antibody to this protein gave a single precipitin band by immunodiffusion and immunoelectrophoresis in agar with the purified protein. This antibody ultrastructurally immunolocalized specifically over tonofilaments in all layers of the epidermis, but showed no reaction in the dermis. The synthesis of this protein in vitro was inhibited by puromycin but not by actinomycin D, suggesting ribosomal synthesis involving a relatively long lived messenger.     

Chromosomal localization of the met proto-oncogene in the mouse and cat genome

The met proto-oncogene was mapped in the mouse and cat genomes with the use of mouse X hamster and cat X rodent somatic cell hybrid DNA panels. Based on these analyses we assigned the met gene to mouse chromosome 6 and to cat chromosome A2. We also assigned the cat raf-1 proto-oncogene to the A2 chromosome; met and raf-1 are the first cloned DNAs mapped to this linkage group. Using an interspecies backcross we further localized met on mouse chromosome 6 to a position proximal to the beta chain of the T-cell receptor. This places met near the obese locus in a region of mouse chromosome 6 that appears to be homologous with the long arm of human chromosome 7. The close linkage of met to the gene responsible for cystic fibrosis in humans suggests that further genetic analysis of mouse chromosome 6 may be useful in developing a mouse model for the disease.     

Chromosomal polymorphism caused by supernumerary chromosomes in the field mouse,Apodemus giliacus

Chromosome studies were made on 74 animals of the field mouse, Apodemus giliacus, a new form of the genus Apodemus described by Kobayashi and Hayata (1970). Extreme variations in number and morphology of chromosomes was observed. While the diploid numbers varied from 48 to 61, the number of acrocentric elements was consistently 48, except for one specimen which had 40 such elements. In contrast, the number and constitution of several biarmed elements and microchromosomes were highly variable in the complement, and, hence, responsible for the polymorphism. Karyotype analysis of meiotic chromosomes, on the basis of Giemsaand quinacrine-stained preparations, some familial studies and breeding experiments revealed that variable elements were supernumeraries of a hitherto undescribed type, whereas the 48 acrocentrics seemed to represent regular autosomes and sex elements. Most of the supernumeraries did not show pairing at metaphase I, but some did form bivalents. Usually, the supernumerary biarmed chromosomes were of regular size and fluoresced rather brightly over their entire length, suggesting that they were heterochromatic in nature. Considering the above findings and other relevant data of some allied species, the differentiation between A. giliacus and A. speciosus might have occurred rather recently, when the former species might have been involved in rapid and divergent chromosomal evolution.Contributions from the Chromosome Research Unit, Sapporo, Japan.     

Coding sequence, genomic organization, chromosomal localization, and expression pattern of the signalosome component Cops2: the mouse homologue of Drosophila alien

The Drosophila alien gene is highly homologous to the human thyroid receptor interacting protein, TRIP15/COPS2, which is a component of the recently identified signalosome protein complex. We identified the mouse homologue of Drosophila alien through homology searches of the EST database. We found that the mouse cDNA encodes a predicted 443-amino-acid protein, which migrates at approximately 50 kDa. The gene for the mouse alien homologue, named Cops2, includes 12 coding exons spanning approximately 30 kb of genomic DNA on the central portion of mouse chromosome 2. Mouse Cops2 is widely expressed in embryonic, fetal, and adult tissues beginning as early as E7.5. Mouse Cops2 cDNA hybridizes to two mRNA bands in all tissues at approximately 2.3 and approximately 4 kb, with an additional approximately 1.9-kb band in liver. Immunostaining of native and epitope tagged proteins localized the mouse Cops2 protein in both the cytoplasm and the nucleus, with larger amounts in the nucleus in some cells.     

Characterization of RT6 bearing rat lymphocytes. I. Ontogeny of the RT6+ subset

The RT6 alloantigen is present on approximately 70% of peripheral T cells in the rat, but is absent from thymocytes and bone marrow lymphocytes. The results of further phenotypic analysis in the present study demonstrated that the RT6 alloantigen is expressed on approximately 45% of the helper/inducer (CD4; W3/25+) and 80% of the cytotoxic/suppressor (CD8; OX8+) peripheral T-cell subsets. Ontogenetic and thymus ablation studies indicated that the RT6+ T-cell subset is thymus-dependent and normally develops after the appearance of RT6-T cells in neonatal rats, and that the expression of RT6 is a post-thymic maturational event. Furthermore, intrathymic adoptive transfer of bone marrow cells demonstrated that RT6+ T cells are thymus-derived cells. These results show that most if not all RT6+ T cells are the progeny of RT6- T cells. However, they do not exclude the possibility that a separate lineage of RT6- T cells exists, which also has OX8+ and W3/25+ subsets. The possible developmental and functional relationships of RT6- and RT6+ T cells in the rat are discussed.     

Fudenine, a C-terminal truncated rat homologue of mouse prominin, is blood glucose-regulated and can up-regulate the expression of GAPDH

Messenger RNA differential display was applied to screen for the blood glucose-regulated genes in SD rat skeletal muscle. The rat homologue of the mouse prominin was thus identified. Comparing to its mouse and human homologues, fudenine was C-terminal truncated due to a single nucleotide deletion. However, its mitochondrial energy transfer signature peptide PQDLVKKLI remained intact. Fudenine, an 592-amino acid containing, 66-kDa glycoprotein, is a novel plasma membrane protein with four transmembrane segments flanking by two large glycosylated extracellular domains. Although it is devoid of the last transmembrane domain comparing to its homologues, fudenine also locates in cell membrane by transfection of fusion plasmid pFudenine-EGFP into CBRH7919 cell and L-6TG cell. Overexpression of fudenine in CBRH7919 cell line up-regulated the mRNA level of GAPDH (3-phosphate glyceraldehyde dehydrogenase), while long-term glucose exposure resulted to reduced GAPDH expression. Since high blood glucose level induced the expression of fudenine in skeletal muscle, which in turn up-regulated the expression of GAPDH, we propose that fudenine might be a candidate gene for diabetes mellitus.