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M J Tymms

Publications and source records attributed to M J Tymms.

At least 19 recordsLinked to original sources
PubMed1997

A novel epithelial-expressed ETS gene, ELF3: human and murine cDNA sequences, murine genomic organization, human mapping to 1q32.2 and expression in tissues and cancer.

The ETS family of genes are implicated in cancers such as Ewings sarcoma, acute myeloid leukemia and chronic myelomonocytic leukemia. Further, they have important functions in embryonic development. Hence, identification and characterization of members of this family are important. We identify a novel ETS family member, ELF3, and report its human and murine cDNA sequences. The mouse cDNA has an alternatively spliced transcript with an extra 60 bp inserted. Hence we present the organization of the murine Elf3 gene together with its exon/intron structure. This gene consists of 9 exons and 8 introns spanning 4.8 kb. ELF3 binds and transactivates ETS sequences and interestingly also shows the ability to bind a GGAT-like purine core, a preferential ETS1/ETS2 type binding site. The expression of ELF3, unlike most other ETS family members, is absent in hematopoietic cells and hematopoietic organs in humans and mice. Intriguingly, the gene is specifically expressed in cell lines of epithelial origin and in organs such as lung, stomach, intestine, kidney that have specialized epithelial cells. We localize the human gene to 1q32.2, a region that is amplified in epithelial tumors of the breast, lung and prostate. Finally, we show that ELF3 expression is increased in a lung carcinoma and adenocarcinoma, as compared to normal tissue. ELF3 is also expressed in cell lines derived from lung cancers. These results suggest that this novel ETS gene may be involved in lung tumorigenesis.

Amino Acid Sequence↗
PubMed1997

A type I interferon signaling factor, ISF21, encoded on chromosome 21 is distinct from receptor components and their down-regulation and Is necessary for transcriptional activation of interferon-regulated genes.

The type I interferons (IFNs) are a family of cytokines, comprising at least 17 subtypes, which exert pleiotropic actions by interaction with a multi-component cell surface receptor and at least one well characterized signal transduction pathway involving JAK/STAT (Janus kinase/signal transducer and activator of transcription) proteins. In a previous report, we showed that a signaling factor, encoded by a gene located on the distal portion of chromosome 21, distinct from the IFNAR-1 receptor, was necessary for 2'-5'-oligoadenylate synthetase activity and antiviral responses, but not for high affinity ligand binding. In the present studies using hybrid Chinese hamster ovary cell lines containing portions of human chromosome 21, we show that the type I IFN signaling molecule, designated herein as ISF21, is distinct from the second receptor component, IFNAR-2, which is expressed in signaling and non-signaling cell lines. The location of the gene encoding ISF21 is narrowed to a region between the 10;21 and the r21 breakpoints, importantly eliminating the Mx gene located at 21q22.3 (the product of which is involved in IFN-induced antiviral responses) as a candidate for the signaling factor. To characterize the action of this factor in the type I IFN signaling pathway, we show that it acts independently of receptor down-regulation following ligand binding, both of which occur equally in the presence or absence of the factor. In addition, we demonstrate that ISF21 is necessary for transcriptional activation of 2'-5'-oligoadenylate synthetase, 6-16, and guanylate-binding protein gene promoter reporter constructs, which are mediated by several signaling pathways. ISF21 represents a novel factor as the localization to chromosome 21, and the data presented in this study exclude any of the known type I IFN signal-transducing molecules.

Animals↗
PubMed1997

ETS1, NFkappaB and AP1 synergistically transactivate the human GM-CSF promoter.

Activation of helper T cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony stimulating factor (GM-CSF) is one such cytokine, whose increased expression results mostly from increases in transcription. Cis-acting elements with NFkappaB, AP1 and ETS-like binding motifs have been identified in the promoter region of the GM-CSF gene, and are important or essential for transcriptional activity following T cell activation. ETS1 is a transcription factor of the ETS family that is expressed in T cells. We have previously shown that ETS1 can transactivate GM-CSF in Jurkat T cells, but only after the cells have been stimulated by treatment with PMA and ionomycin, agents that mimic T cell activation. Thus we proposed that ETS1, which is expressed constitutively in Jurkat cells, may act in concert with PMA/ionomycin inducible factors. Here we show that ETS1 can transactivate a GM-CSF reporter construct in unstimulated Jurkat cells, providing that either NFkappaB or AP1 transcription factors are supplied by co-transfection. We confirm that binding of endogenous NFkappaB and AP1 is induced following PMA/ionomycin treatment of T cells. Transactivation by ETS1, NFkappaB and AP1 is synergistic, and mutation of the individual binding sites reveals that the transcriptional activities of these factors are interdependent. Our results suggest that constitutive ETS1, and inducible NFkappaB and AP1, cooperate as part of a higher order transcriptional complex in activated T cells.

Animals↗
PubMed1997

Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG methylation.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine spermatogenesis-specific isoform of the E1a subunit of the pyruvate dehydrogenase complex. To begin to delineate the mechanisms regulating its expression in vivo, we have generated transgenic mice lines carrying Pdha-2 promoter deletion constructs. Here we report that transgenic mice harboring a construct containing only 187 bp of promoter and upstream sequences (core promoter) is sufficient for directing the testis-specific expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Like the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. Our studies also show a correlation between CpG methylation within the core promoter and its capacity to regulate transcription. In NIH 3T3 cell lines stably transfected with the Pdha-2 core promoter-CAT construct, high levels of CAT reporter expression are observed, whereas the endogenous Pdha-2 gene is repressed. In these cells, the CpG dinucleotides residing within the transfected promoter are hypomethylated whereas those residing in the endogenous promoter are methylated. Furthermore, promoter activity can be abated by the in vitro methylation of its CpG dinucleotides. DNase I footprint analysis indicates that at least one site for the methylation-mediated repression may occur through the ATF/cyclic AMP response element binding element located within the core promoter. Mutations within this element reduces activity to approximately 50% of the wild-type promoter activity. These results suggest that tissue-specific gene expression may be modulated by other mechanisms in addition to specific transcription factor availability and cooperativity. We propose that methylation may be a mechanism by which repression of the testis-specific Pdha-2 gene is established in somatic tissue.

3T3 Cells↗
PubMed1996

Down's syndrome-like skeletal abnormalities in Ets2 transgenic mice.

Expression of Ets2, a proto-oncogene and transcription factor, occurs in a variety of cell types. During murine development it is highly expressed in newly forming cartilage, including in the skull precursor cells and vertebral primordia. Ets2 is located on human chromosome 21 (ref. 8) and is overexpressed in Down's syndrome (trisomy 21). Here we generate transgenic mice to investigate the consequences of overexpression of Ets2. We find that mice with less than 2-fold Ets2 overexpression in particular organs develop neurocranial, viscerocranial and cervical skeletal abnormalities. These abnormalities have similarities with the skeletal anomalies found in trisomy-16 mice and humans with Down's syndrome, in which the gene dosage of Ets2 is increased. Our results indicate that Ets2 has a role in skeletal development and implicate the overexpression of Ets2 in the genesis of some skeletal abnormalities that occur in Down's syndrome.

Abnormalities, Multiple↗
PubMed1996

Construction of a mouse blastocyst cDNA library by PCR amplification from total RNA.

Studies of the development and differentiation of early mammalian embryos have been severely limited by the paucity of material. Such studies have been largely restricted to the examination of abundant genes/proteins or to developmental expression studies of known genes for which DNA sequence data are available, allowing the use of reverse transcription and polymerase chain reaction amplification (RT-PCR). To eliminate the need for hundreds or thousands of oocytes or embryos in the construction of representative cDNA libraries, we describe a technique for generating and cloning cDNA using small caesium chloride gradient centrifugation to isolate total RNA from oocytes or embryos, followed by RT-PCR of mRNA from this total RNA. Total RNA was isolated from 70 mouse blastocysts. A portion of the cDNA generated (equivalent to seven blastocysts) was cloned, yielding a mouse blastocyst cDNA library of 1 million clones. We show that the library is representative in that it contains beta-actin, intracisternal A-type particles, tissue plasminogen activator, and B1 and B2 repetitive elements in frequencies comparable with published data from conventionally constructed libraries and estimates of mRNA abundance from expression studies. Furthermore, DNA sequencing of 22 clones chosen at random and compared with DNA sequence databases shows that approximately half are novel sequences. These data demonstrate that representative cDNA libraries can be constructed in situations where cell numbers are limiting and will facilitate the isolation of novel and interesting clones.

Animals↗
PubMed1995

A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses.

To examine the in vivo role(s) of type I interferons (IFNs) and to determine the role of a component of the type I IFN receptor (IFNAR1) in mediating responses to these IFNs, we generated mice with a null mutation (-/-) in the IFNAR1 gene. Despite compelling evidence for modulation of cell proliferation and differentiation by type I IFNs, there were no gross signs of abnormal fetal development or morphological changes in adult IFNAR1-/- mice. However, abnormalities of hemopoietic cells were detected in IFNAR1 -/- mice. Elevated levels of myeloid lineage cells were detected in peripheral blood and bone marrow by staining with Mac-1 and Gr-1 antibodies. Furthermore, bone marrow macrophages from IFNAR1 -/- mice showed abnormal responses to colony-stimulating factor 1 and lipopolysaccharide. IFNAR1 -/- mice were highly susceptible to viral infection: viral titers were undetected 24 hr after infection of IFNAR1 +/+ mice but were extremely high in organs of IFNAR1 -/- mice, demonstrating that the type I IFN system is a major acute antiviral defence. In cell lines derived from IFNAR1 -/- mice, there was no signaling in response to IFN-alpha or -beta as measured by induction of 2'-5' oligoadenylate synthetase, antiviral, or antiproliferative responses. Importantly, these studies demonstrate that type I IFNs function in the development and responses of myeloid lineage cells, particularly macrophages, and that the IFNAR1 receptor component is essential for antiproliferative and antiviral responses to IFN-alpha and -beta.

Animals↗
PubMed1995

ETS1 transactivates the human GM-CSF promoter in Jurkat T cells stimulated with PMA and ionomycin.

Activation of T helper cells results in coordinate expression of a number of cytokines involved in differentiation, proliferation and activation of the haematopoietic system. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one such cytokine whose increased expression results partly from increases in transcription. Cis-acting elements with NF kappa B, AP-1 and ETS-like motifs have been identified in the promoter region of the GM-CSF gene, which are important for transcriptional activity following PMA and ionomycin stimulation. A number of the ETS family of transcription factors are expressed in T cells, including ETS1 and ELF1. Here we describe the ability of these factors to interact with a site (GM5), located within the CLE0 element, -47 to -40 upstream of the GM-CSF transcription initiation site. Exogenous ETS1, but not ELF1, can transactivate GM-CSF, through the GM5 site, in a PMA/ionomycin dependent manner. Other unidentified ETS-like factors present in Jurkat cells are also capable of binding GM5. Mutation of the core ETS binding site from -GGAA- to -GGAT- prevents the binding of ETS-like factors with the exception of ETS1. The GM-CSF promoter, modified in this way to be ETS1 specific, is fully responsive to PMA/ionomycin induction, in addition to ETS1 transactivation in the presence of PMA and ionomycin. Together these data suggest that ETS1 may be involved in mediating the increased GM-CSF production associated with T cell activation.

Base Sequence↗
PubMed1995

Human ERG is a proto-oncogene with mitogenic and transforming activity.

The ETS related gene, ERG, is one of 20 or more genes belonging to the ETS family of transcription factors. Translocation of the ERG gene t(21;22) results in the chimeric fusion transcript seen in approximately 10% of Ewings sarcomas. In addition, recent studies have shown that a reciprocal translocation t(21;16) of ERG gives rise to two aberrant transcripts seen in some forms of acute myeloid leukaemia. In vitro studies have linked the up regulation of ERG expression with stromal cell independence in erythroleukemic clones and shown that the ERG related genes ETS1 and ETS2 have a mitogenic and transforming activity when overexpressed in NIH3T3 cells. Interestingly ERGB/FLI-1, which is also involved in Ewings sarcoma translocations and shares a very high sequence identify with ERG has been reported to be unable to transform NIH3T3 cells. In this study we investigate the effects of overexpression of ERG on cell proliferation, factor dependence, growth in soft agar and tumorigenesis in nude mice. An ERG expression construct with the human ERG2 cDNA driven by the sheep metallothionein la promoter (sMTERG) was transfected into NIH3T3 cells. Clonal cell lines overexpressing ERG were established. The cell lines became morphologically altered, grew in low serum and serum free media and gave rise to colonies in soft agar suspension. Furthermore, we demonstrate that after subcutaneous injection these clones grow as solid tumors in nude mice. These data demonstrate that c-ERG is a proto-oncogene capable of transforming NIH3T3 cells. Therefore, overexpression or inappropriate expression of ERG may contribute to oncogenesis.

3T3 Cells↗
PubMed1994

A gene on human chromosome 21 located in the region 21q22.2 to 21q22.3 encodes a factor necessary for signal transduction and antiviral response to type I interferons.

The type I interferons (IFNs) are a family of multifunctional cytokines which includes the 15 IFN alpha subtypes and IFN beta. These IFNs compete for binding to cell surface receptors. However, murine cells transfected with a cDNA for a human IFN alpha receptor (IFNAR) developed an antiviral response only to human IFN alpha B, but not to human IFN alpha 2 nor -beta(1). In this study we show, using a panel of CHO-human chromosome 21 hybrid cell lines which all express IFNAR, that only those containing the region 21q22.2 to 21q22.3 transduce signals for IFN responses. Two such hybrid cell lines responded to IFNs alpha 2, -alpha B and -beta by induction of 2'-5' oligoadenylate synthetase and resistance to viral infection. Other hybrid cell lines, that lacked the region 21q22.2-3, failed to transduce signals as above; even though they expressed IFNAR and bound human IFN alpha 2, -alpha B, and -beta. These data demonstrate that a gene(s) located in the region 21q22.2-3 encodes a factor(s) which is necessary for signaling but does not influence ligand binding. This factor is not the cofactor required for IFN gamma signaling which is located in the region 21p to 21q22.1(2).

2',5'-Oligoadenylate Synthetase↗
PubMed1994

Regulation of gene expression by transcription factors Ets-1 and Ets-2.

The ETS family of transcription factors have a DNA-binding domain in common that binds a core GGA(A/T) DNA sequence. A large number of proteins have now been identified that contain an ETS DNA-binding domain (see review by Wasylyk et al., 1993). Ets-1 was first described as the cellular homolog of v-ets. which is translated as a 135-kDa gag-myb-ets fusion protein from the replication-deficient retrovirus E26 in chickens. Ets-2 was subsequently described as a closely related protein that contains the highly conserved ETS DNA-binding domain. This paper considers the manner by which the two closely related genes, Ets-1 and Ets-2, apparently play distinct roles in embryo development and in the immune system of adult mice. Although both Ets-1 and Ets-2 transform fibroblasts (Seth et al., 1989), the temporal and tissue-specific expression patterns suggest that these two proteins play distinct biological roles and consequently transactivate different downstream cellular target genes.

Amino Acid Sequence↗
PubMed1994

Mouse testis Pdha-2 promoter upstream sequences confer tissue-and temporal-specific activity in transgenic mice.

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine testis-specific isoform of the E1 alpha subunit of the pyruvate dehydrogenase complex. To elucidate the mechanisms regulating its expression in vivo, we have begun to investigate the Pdha-2 promoter in transgenic mice. In this paper, a construct containing 3.0 kb of promoter and upstream sequences is reported to be sufficient for directing the testis-specific expression of a CAT reporter gene in mice harbouring the transgene. Similarly to the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. However, the 3.0-kb Pdha-2 promoter is not active in somatic tissue suggesting that repressor elements may be present within these sequences.

3T3 Cells↗
PubMed1994

Expression of copper/zinc superoxide dismutase and glutathione peroxidase in organs of developing mouse embryos, fetuses, and neonates.

The rise in antioxidant enzyme activity in the lungs of late-gestation fetuses is thought to be caused by the preparation of the pulmonary antioxidant system for birth. However, recent data have shown that such a rise also occurs in the livers of late-gestation fetuses. Consequently, this surge cannot solely be ascribed to the preparation of the pulmonary antioxidant system for birth. In this study we examine the expression of copper/zinc superoxide dismutase (Sod1) and glutathione peroxidase (Gpx1) in various organs of late-gestational mouse fetuses. Furthermore, we compare the expression of these genes in organs of fetuses, neonates, and adult mice. These studies were carried out to investigate whether the change in mRNA levels for these two genes is related to a developmental change in oxidant stress. Our data demonstrate that an increase in both Sod1 and Gpx1 mRNA occurs in lungs and livers of late-gestational mouse fetuses. The brain demonstrates an increase in Sod1 expression at or around the time of birth, the kidney shows an elevation in Gpx1 mRNA levels, and the heart fails to demonstrate a surge in both Sod1 and Gpx1 mRNA levels. Our data show that the liver is the organ with the highest levels of Sod1 and Gpx1 mRNA in embryos and neonates (immediately after birth). In the adult, the liver has the highest levels of Sod1 mRNA and the spleen the highest level of Gpx1 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Structure-function study of the region encompassing residues 26-40 of human interferon-alpha 4: identification of residues important for antiviral and antiproliferative activities.

The highly conserved amino acid residues Leu-30 and Arg-33 of human interferon-alpha 4 (IFN-alpha 4) have previously been identified as important for biological activity. In this study, the sequence around Arg-33 was targeted to determine the importance of other residues in this region. A library of analogues containing amino acid substitutions spanning residues 26-37 was generated using site-directed and random mutagenesis. Analogues were expressed in vitro and assayed for antiviral and antiproliferative activity on human cells. No significant separation between the antiviral and antiproliferative activities was observed for any of the analogues tested. Substitutions at positions 26, 27, 31, 32, 34, 35, and 37, did not substantially affect biological activity. However, substitution of Phe-36 with arginine resulted in a greater than 100-fold decrease in biological activity. Thus, together with previous data, the residues in this region identified as most important for biological activity include Leu-30, Arg-33, and Phe-36. Recently published models for the three-dimensional structure of human IFN-alpha and the X-ray crystallographic structure of murine IFN-beta, suggest that the region investigated in this study forms a loop at the surface of the protein. Thus, residues Leu-30, Arg-33, and Phe-36, could be involved in binding to the Type-I IFN receptor, or in interactions with signal-transducing molecules.

Base Sequence↗
PubMed1991

Structure-function studies of human interferons-alpha: enhanced activity on human and murine cells.

To identify functionally important regions of the human interferon (IFN)-alpha molecule, mutagenesis in vitro of human IFN-a genes was used to create analogs with deletions or specific amino acid replacements. These analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate protein synthesis system. Deletion of 7 highly conserved hydrophilic amino acids from the C-terminus of human IFN-alpha 4 reduced, but did not abolish, antiviral activity on human cells. However, analogs with deletions of 15 or 25 amino acids from the C-terminus, or 28 amino acids from the N-terminus, had no measurable antiviral activity. The antiviral activity of human IFN-alpha 4 was increased by substitution of cysteine for serine at position 86, and lysine for arginine at position 121. However, other amino acid substitutions at positions 121, 122 or 123 reduced antiviral activity. The size of the side chain of the amino acid residue at position 130 was shown to be important. Replacement of the absolutely conserved leucine residue at position 131 with glutamine had little effect on antiviral activity. However, the introduction of a proline residue at this position abolished antiviral activity, probably due to the formation of a beta turn in the polypeptide chain. The antiviral activity of human IFN-alpha 4 on murine cells was increased by substitutions at positions 86, 121 and 133. This study illustrates the utility of the in vitro mutagenesis and rabbit reticulocyte lysate systems for the investigation of structure-function relationships, and extends our knowledge of the biologically active regions and species specificity of the human IFN-alpha molecule.

Animals↗
PubMed1991

Universal antibodies to human interferon-alpha subtypes--the production of antipeptide antibodies to conserved regions of interferon-alpha.

Antibodies to three conserved regions of all human interferon (IFN)-alpha 1 subtypes were raised by immunizing rabbits with short synthetic peptides coupled to a carrier. These peptides correspond to amino acid residues 29-36, 31-36, 126-131, 139-151, and 142-151 of the consensus sequence of IFN-alpha 1. The antibodies were tested for reactivity with IFN-alpha 1, -alpha 2a, -alpha 2b, -alpha 4a, and -alpha 14 and IFN-beta. Most antipeptide antibodies react only weakly with IFN-alpha subtypes; only the antibodies raised against the peptide corresponding to residues 142-151 conjugated to keyhole limpet hemocyanin using glutaraldehyde react appreciably with all IFN-alpha subtypes tested. These antipeptide antibodies are potentially universal antibodies to the human IFN-alpha 1 subtypes, since they exhibit similar binding affinity to all the IFN-alpha subtypes tested and do not react with IFN-beta.

Amino Acid Sequence↗