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T S Papas

Publications and source records attributed to T S Papas.

At least 73 records · Page 4Linked to original sources
PubMed1991

The human ETS1 gene: genomic structure, promoter characterization and alternative splicing.

Genomic clones encompassing the human ETS1 gene were isolated and utilized to define its molecular organization. This gene consists of eight exons spanning over 60 kb. The 5' end of the human ETS1 gene was subcloned and characterized. S1 nuclease, primer extension and RNAase protection analyses of human mRNAs showed multiple transcription initiation sites. DNA sequence analysis indicated a high G + C content in the promoter region and the absence of either a 'TATA' box or a 'CAAT' box. Six consensus recognition sequences for the transcription factor SP1, two AP1 consensus sequences and one consensus AP2 recognition sequence were identified, as well as two GC elements with dyad symmetry. A palindromic region similar to the serum response element of the c-fos gene and two octamer consensus recognition sequences were located upstream of the promoter region. A series of promoter deletion constructs positioned upstream from the bacterial chloramphenicol acetyl transferase gene were transfected into HeLa cells and their functional promoter activity assayed. The deletion constructs identified the 5' boundary for maximum promoter activity at 486 bp upstream of the first initiation site and suggest possible positive and negative regulatory regions. Polymerase chain reaction analysis of ETS1 cDNA identified several amplified products, indicating alternative splicing. In addition to the presence of mRNA products lacking exon VII, products lacking exon IV, as well as ones lacking both exons IV and VII, were found.

Base Sequence↗
PubMed1990

Requirement of ets-2 expression for Xenopus oocyte maturation.

A molecular clone of the Xenopus laevis ets-2 gene was isolated from an oocyte complementary DNA library. The amount of messenger RNA (mRNA) in each oocyte or embryo was almost constant during oogenesis and was maintained until the blastula stage of embryonic development, indicating that the observed 3.2-kilobase transcript is a maternal message. The only normal adult tissue in which ets-2 mRNA was detected was the ovary. Injection of antisense oligonucleotides homologous to the ets-2 sequence into oocytes led to degradation of the mRNA and blocked hormone-induced germinal vesicle breakdown. The ets-2 product is thus required for the meiotic maturation of Xenopus oocytes.

Animals↗
PubMed1990

Sequence-specific binding of human Ets-1 to the T cell receptor alpha gene enhancer.

Expression of the human T cell receptor (TCR) alpha gene is regulated by a T cell-specific transcriptional enhancer that is located 4.5 kilobases (kb) 3' to the C alpha gene segment. The core enhancer contains two nuclear protein binding sites, T alpha 1 and T alpha 2, which are essential for full enhancer activity. T alpha 1 contains a consensus cyclic adenosine monophosphate (cAMP) response element (CRE) and binds a set of ubiquitously expressed CRE binding proteins. In contrast, the transcription factors that interact with the T alpha 2 site have not been defined. In this report, a lambda gt11 expression protocol was used to isolate a complementary DNA (cDNA) that programs the expression of a T alpha 2 binding protein. DNA sequence analysis demonstrated that this clone encodes the human ets-1 proto-oncogene. Lysogen extracts produced with this cDNA clone contained a beta-galactosidase-Ets-1 fusion protein that bound specifically to a synthetic T alpha 2 oligonucleotide. The Ets-1 binding site was localized to a 17-base pair (bp) region from the 3' end of T alpha 2. Mutation of five nucleotides within this sequence abolished both Ets-1 binding and the activity of the TCR alpha enhancer in T cells. These results demonstrate that Ets-1 binds in a sequence-specific fashion to the human TCR alpha enhancer and suggest that this developmentally regulated proto-oncogene functions in regulating TCR alpha gene expression.

Base Sequence↗
PubMed1990

Oncogenes in hematopoietic and hepatic fish neoplasms.

Neoplastic transformation of cells has often been associated with changes in cellular oncogenes. While much information has been collected in mammalian systems, relatively little is known about the molecular basis of tumor progression in lower vertebrates. For our studies, tumors were collected from feral northern pike (Esox lucius) from Ostego Lake, MI, where the local population exhibited a 15% incidence of large external lymphomas. In laboratory studies, tumors were induced under controlled conditions by known mammalian carcinogens in the Japanese medaka (Oryzias latipes), a small aquarium fish widely used in carcinogenicity studies. DNA isolated from these tumors was assayed for transforming sequences by transfection into NIH3T3 cells. DNAs from the northern pike lymphomas and the chemically induced tumors in the medaka were able to transform NIH3T3 cells and induce tumors in athymic mice. The results of our studies to date are summarized here, together with the current status of oncogene activation in other fish systems.

Animals↗
PubMed1990

ETS family of genes in leukemia and Down syndrome.

The human ETS2 and ERG genes are members of the ETS gene family, with sequence homology to the viral ets gene of the avian erythroblastosis retrovirus, E26. These genes are located on chromosome 21 and molecular genetic analysis of Down syndrome (DS) patients with partial trisomy 21 suggested that ETS2 may be a gene within the minimal DS genetic region. We have, in fact, been able to confirm the presence of the ETS2 gene dosage in triplicate occurring in occult human 21 chromosome abnormalities. It is known that ERG and ETS2 gene translocations occur in certain specific leukemias associated with defined chromosome rearrangements [e.g., t(8;21)]. Moreover, it is known that DS individuals are at greater risk for leukemic disease than their normal familial cohorts, implying that trisomy of that region of human chromosome 21 may play a role in the development of this type of neoplasia. The human ETS genes, first identified in our laboratory, are highly conserved, being found from lower organisms, like Drosophila and sea urchin, to humans. In mammals, the ETS genes are structurally distinct, located on separate chromosomes; they are transcriptionally active and differentially regulated. The ETS2 protein is phosphorylated and turns over with a half-life of approximately 20 min. After activation with the tumor promoter, TPA, the level of ETS2 elevates 5- to 20-fold. The properties of the ETS2 protein, such as nuclear localization, phosphorylation, rapid turnover, and response to protein kinase C, indicate that this protein belongs to a group of oncogene proteins thought to have regulatory functions in the nucleus. In the mouse thymus ets-1 and ets-2 are 8-10-fold higher, respectively, in the CD4+ subset than in other subsets examined, suggesting a role in T-cell development for these genes. Cells transfected with the cellular ets-2 gene, expressing higher levels of ets-2 products, showed a stimulated proliferation response, abolished their serum requirement and formed colonies in soft agar that could induce tumors in nude mice. Collectively, these data suggest that this family of genes might play a role in controlling specific steps of the signaling transduction pathway. Thus, the ETS genes, as other genes with homology to viral oncogenes, might be instrumental in regulating cellular growth and differentiation, as well as organismal development.

Animals↗
PubMed1990

Heterogeneity of Nef proteins in cells infected with human immunodeficiency virus type 1.

Human T-lymphocytic cell line H9 infected with the HTLV-IIIB isolate of human immunodeficiency virus type 1 (HIV-1) synthesizes two forms of the Nef protein (p25 and p27) that differ both in molecular weight and charge. Different subpopulations of viruses were isolated from the HTLV-IIIB stock which induce expression of only p25 or p27. Cells infected with HIV-1 derived from the HXB3 clone of the HTLV-IIIB isolate made only the p25 species, whereas the 8E5/LAV cell line which harbors a single defective LAV provirus produces only the p27 species. These findings are consistent with the notion that the HTLV-IIIB isolate consists of at least two distinct variants with different nef genes, one specifying p25 and the other encoding p27. After a considerable number of passages in culture, H9 cells chronically infected with the HTLV-IIIB isolate produced high levels of p25 and lower levels of p27. Passages in culture appear to select for a subpopulation of virus variants that specify high levels of p25 Nef expression.

Cell Line↗
PubMed1990

Subtraction hybridization cDNA libraries from colon carcinoma and hepatic cancer.

cDNA clones of differentially expressed mRNAs in a colon carcinoma and a hepatocellular carcinoma have been isolated by subtractive cDNA cloning. The subtracted material is at least 90 X enriched for differentially expressed sequences and can be used for construction of subtractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. Commercially available lambda ZAP II is used for construction of primary libraries since single-stranded phage bearing the cloned cDNA can be excised in vivo and because lambda libraries are convenient for subsequent screening and manipulations. Rare mRNAs (less than 0.01% abundance), which are differentially expressed, can be isolated utilizing this procedure.

Bacteriophage lambda↗
PubMed1990

Reciprocal expression of human ETS1 and ETS2 genes during T-cell activation: regulatory role for the protooncogene ETS1.

The expression of the protooncogenes ETS1 and ETS2 has been studied in purified human T cells activated either by cross-linking of the T-cell receptor-CD3 complex on their cell surface or by direct stimulation with phorbol esters and ionomycin. Our results show that resting T cells express high levels of ETS1 mRNA and protein, while expression of ETS2 is undetectable. Upon T-cell activation, ETS2 mRNA and proteins are induced, while ETS1 gene expression decreases to very low levels. Late after stimulation, ETS1 mRNA is reinduced and maintained at a high level, while ETS2 gene expression decreases to undetectable levels. Therefore, it appears that in human T cells, ETS2 gene products are associated with cellular activation and proliferation, while ETS1 gene products are preferentially expressed in a quiescent state.

Calcium↗
PubMed1990

Molecular and functional characterization of the promoter of ETS2, the human c-ets-2 gene.

The 5' end of the human c-ets-2 gene, ETS2, was cloned and characterized. The major transcription initiation start sites were identified, and the pertinent sequences surrounding the ETS2 promoter were determined. The promoter region of ETS2 does not possess typical "TATA" and "CAAT" elements. However, this promoter contains several repeat regions, as well as two consensus AP2 binding sites and three putative Sp1 sites. There is also a palindromic region similar to the serum response element of the c-fos gene, located 1400 base pairs (bp) upstream from the first major transcription initiation site. A G + C-rich sequence (GC element) with dyad symmetry can be seen in the ETS2 promoter, immediately following an unusually long (approximately 250-bp) polypurine-polypyrimidine tract. A series of deletion fragments from the putative promoter region were ligated in front of the bacterial chloramphenicol acetyltransferase gene and tested for activity following transfection into HeLa cells. The 5' boundary of the region needed for maximum promoter activity was found to be 159 bp upstream of the major initiation site. This region of 159 bp contains putative binding sites for transcription factors Sp1 and AP2 (one for each), the GC element, one small forward repeat, one inverted repeat, and half of the polypurine-pyrimidine tract. The promoter of ETS2 (within the polypyrimidine tract) serves to illustrate an alternative structure that may be present in genes with "TATA-less" promoters.

Base Sequence↗
PubMed1990

Bacterially produced HIV-2 env polypeptides specific for distinguishing HIV-2 from HIV-1 infections.

Five unique recombinant polypeptides, each encoded by a DNA segment representing a different region of the HIV-2 (NIH-Z strain) env gene, were produced at relatively high levels (greater than or equal to 5%) as cII-fusion products in Escherichia coli. These recombinant polypeptides were characterized serologically by the Western blot assay against a panel of HIV-2 and HIV-1 antibody-positive sera, and with normal human sera (HIV-1 and HIV-2 antibody negative). Only those polypeptides that are encoded by a segment of the env gene from the N-terminal region of the transmembrane protein gp35 (amino acids 537 to 707) were immunoreactive. Three polypeptides (921, 996, and 997), each encoding this immunoreactive region of the HIV-2 (NIH-Z) gp35, reacted strongly and specifically with antibodies in sera from HIV-2-positive individuals, but not with antibodies in sera from HIV-1-positive or HIV-uninfected individuals. These results show that the N-terminal region of the HIV-2 gp35 contains a highly antigenic determinant which is strongly immunogenic in HIV-2-infected individuals. The gp35-encoded recombinant env polypeptides can potentially be used in diagnostic assays to specifically differentiate between HIV-2 and HIV-1 infections.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Defining target sequences of DNA-binding proteins by random selection and PCR: determination of the GCN4 binding sequence repertoire.

We developed a simple and accurate method to define the sequence recognition properties of DNA-binding proteins. The method employs polymerase chain reaction (PCR) amplification of sequences selected from a mixture of random oligonucleotides by the gel mobility-shift assay. We used this method to define the sequence requirement of the binding domain of the yeast transcriptional activator GCN4. Using a total of 200 ng of purified protein and four cycles of binding and subsequent amplification, we identified the TGA-(C/G)TCA sequence as the binding consensus of GCN4, which is consistent with the previously reported recognition sequence. In addition, our data indicate that GCN4 can bind with lower affinity to sequences that differ from the optimal sequence in one or even two positions. The most common variation was the C to A at position +2. The majority of the substitutions that still allowed binding were 3' to the central C residue indicating that the two sides of the palindromic recognition sequence are not equivalent.

Base Sequence↗
PubMed1990

Monoclonal antibodies specific to human ETS-2 oncoprotein: recognition of epitopes clustered on the B domain.

Six monoclonal antibodies were prepared from mice immunized with a bacterially expressed human ets-2 protein. These antibodies specifically recognize the two human ets-2-encoded proteins p56 and p54 but failed to react with chicken, mouse, rat, bovine, or monkey proteins, suggesting that the antibodies recognize epitopes specific to the human ets-2 protein. Differential reactivities of these monoclonal antibodies with the peptide fragments generated by partial proteolytic digestion of the bacterially expressed ets-2 protein indicated that the six antibodies recognize at least three distinct epitopes in the B domain of the ets-2 protein. Immunoprecipitation experiments comparing native and denaturing conditions suggested that the ets-2 domain detected by the monoclonal antibodies is masked in the native condition by either protein folding or interacting proteins. The biochemical analysis of the ets-2 protein will be facilitated by the development of these monoclonal antibodies, which may be useful as both domain-specific probes and tools for specifically detecting the human ets-2 protein in heterologous expression systems.

Animals↗
PubMed1990

Phosphorylation of the ETS-2 protein: regulation by the T-cell antigen receptor-CD3 complex.

Phosphorylation of the human ets-2 protein in response to mitogenic signals to T lymphocytes was investigated in Jurkat cells. Activation of the cells by antibodies against the T-cell antigen receptor-CD3 complex or by concanavalin A was followed within 5 min by increased phosphorylation of the protein, as shown by a mobility shift of the protein from 54 to 56 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increased incorporation of 32P. The Ca2+ ionophores A23187 and ionomycin were able to mimic this effect, suggesting that this phosphorylation is mediated by Ca2+.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Molecular analysis of the ets genes and their products.

Organisms from human to Drosophila have been found to contain cellular sequences and transcripts that are homologous to the ets region of the avian retrovirus, E26. Ets-related sequences are present on at least two distinct functional loci in chickens and mammals, and have been designated ets-1 and ets-2. The E26 virus transduced sequences from the chicken ets-1 locus, which encompasses over 60 kb of DNA. The ets genes characterized so far from sea urchin and Drosophila are most closely related to the 3' end of the known ets genes. The predicted viral and avian ets proteins are very similar, except at the termini. The similarity between the predicted ets proteins so far described is discussed. The ets proteins have been identified and localized by immunoprecipitation and immunofluorescence. While the ets-1 proteins are found in the nuclear and cytoplasmic fractions, the viral gag-myb-ets protein (p135) and the ets-2 proteins are nuclear. The ets-1 and ets-2 genes are differentially regulated in different cell types, probably reflecting unique controlling elements. Because chromosomal translocations have been associated with different human leukemias, studies addressing the possible association with the ETS1 (11q23) or ETS2 (21q22.3) loci are reviewed.

Alzheimer Disease↗
PubMed1990

Isoforms of the human ets-1 protein: generation by alternative splicing and differential phosphorylation.

The ets-1 gene belongs to the ets gene family (ets-1, ets-2, erg, and elk) and is homologous to the v-ets oncogene found in the avian leukemia virus E26. The ets-1 gene products were characterized using a specific monoclonal antibody developed against a bacterially expressed v-ets protein. The ets-1 gene product in the human T-cell line CEM was found to consist of at least six species: four major species with apparent molecular weights of 51 kDa (p51), 48 kDa (p48), 42 kDa (p42), and 39 kDa (p39); and two minor species of 52 kDa (pp52) and 49 kDa (pp49), which are demonstrated to be the phosphorylated forms of p51 and p48, respectively. All of the ets-1 proteins are related to each other and are considered products of the ets-1 gene. Subcellular localization showed that the pp52 and p51 are found mainly in the cytoplasm, while p48 and p39 are found mainly in the nucleus. Specific antibodies against various exons of ets-1 showed that both p42 and p39 lack a region corresponding to exon VII. Polymerase chain reaction analyses revealed the presence of an additional RNA product that corresponds to mRNA lacking exon VII. These results suggest that the human ets-1 gene encodes multiple proteins that are generated by at least two distinct mechanisms: alternative splicing of mRNA and protein phosphorylation.

Amino Acid Sequence↗
PubMed1990

The human ETS-2 gene promoter: molecular dissection and nuclease hypersensitivity.

The human ETS-2 gene is a homolog of the v-ets oncogene of the E26 virus, coding for a 56 kilodalton nuclear protein. Herein we characterize the ETS-2 gene promoter, using a series of deletion constructs. Analysis of the nucleotide sequences from -3600 bp to +141 bp reveal that the region -159 bp to +141 bp is absolutely essential for maximum promoter activity. This region includes half of the lengthy polypyrimidine (CT) tract of the ETS-2 promoter, one Sp1 binding site and the GC element proximal to the initiation site. This CT tract is able to increase in an orientation independent manner, the transcription from alpha-globin promoter. Several S1 hypersensitive sites are found to be located in this promoter region, using chromatin and supercoiled DNA, in close proximity with cis regulatory elements. Our results indicate that an unusually long (approximately 250 bp) CT tract is necessary for ETS-2 transcription and this tract can also serve as a transcription activator using a heterologous promoter.

Base Sequence↗