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N K Bhat

Publications and source records attributed to N K Bhat.

13 recordsLinked to original sources
PubMed1992

The basal promoter elements of murine cytochrome c oxidase subunit IV gene consist of tandemly duplicated ets motifs that bind to GABP-related transcription factors.

DNA sequences required for expression of the mouse cytochrome c oxidase subunit IV (COXIV) promoter were identified by transient expression of recombinant COXIV-chloramphenicol acetyltransferase constructs in COS and NIH-3T3 cells. Activity of the COXIV promoter is shown to depend upon upstream Sp1 binding sequences and two tandemly repeated 21-base pair sequence elements each mapping to sites of mRNA initiation. Each initiation region repeat contains a binding site for an ets-related transcription factor which demonstrates specificity for the characteristic GGAA ets sequence motif and reactivity with an ets domain-directed monoclonal pan ets antibody. The two 21-base pair repeats are sufficient for transcriptional activity suggesting that the ets-related factor may be involved in both transcriptional activation and start site positioning. The ets-related protein found in COS nuclear extracts is shown to be identical or closely related to the GA-binding protein (GABP) by comparison of electrophoretic mobilities and immunological reactivities of DNA-protein complexes formed with purified recombinant expressed GABP alpha and beta subunits. Sp1 and the GABP-related factors also bind to another mouse cytochrome oxidase subunit gene COXVb. The similar promoter features of these two genes suggests a possible means of coordinate transcriptional regulation among such respiratory proteins.

3T3 Cells↗
PubMed1992

A positive regulator of the ribosomal protein gene, beta factor, belongs to the ETS oncoprotein family.

The beta factor, which interacts with the rpL32 promoter, binds to the sequence 5'-GAGCCGGAAGTG and trans-activates this gene. Comparison of the DNA sequences bound by the beta factor with those bound by other known DNA-binding proteins revealed that the ETS proteins interact with similar DNA sequences. Consequently we have examined the relationship of the beta factor to the several ETS proteins so far reported. Antibody and oligonucleotide competition experiments, performed by using electrophoretic shift analysis, revealed that the beta factor contains ETS epitopes and that it is immunologically related to both of the GA-binding proteins (GABPs), implying that the beta factor may consist of two separate protein subunits.

Amino Acid Sequence↗
PubMed1992

Human ETS1 oncoprotein. Purification, isoforms, -SH modification, and DNA sequence-specific binding.

The human ETS1 proto-oncogene proteins have been isolated from the T-cell leukemia line, CEM, by immunoaffinity chromatography and their identity confirmed by NH2-terminal amino acid sequencing. Incubation of CEM cells with N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) indicates that ETS proteins can be modified in their cellular context and that pretreatment of the cells with N-ethylmaleimide (NEM) protects ETS1 proteins from TLCK modification. These data show that ETS1 proteins can exist in at least two different states, -SH-available and -SH-protected. Renatured human ETS1 has DNA sequence-specific binding to the PEA3 (CAGGAAGT) motif. The ETS1.PEA3 complex can be observed by electrophoretic mobility shift assays (EMSA). Purified ETS1 retards a band which is exactly the same size as a complex that is retarded from nuclear extracts prepared from CEM cells. Reduced ETS1 is required to form the ETS1.PEA3 complex, however; modification of the ETS1 -SH groups by either NEM or by TLCk does not inhibit formation of the complex. The ETS1.PEA3 complex formed with TLCK-modified ETS1 has a slower mobility than the complex formed with unmodified ETS1. Zone sedimentation analysis of purified ETS1 indicates that it is the monomer of ETS1 which binds to the PEA3 oligonucleotide.

Amino Acid Sequence↗
PubMed1992

Characterization and uses of monoclonal antibody derived against DNA binding domain of the ets family of genes.

A monoclonal antibody recognizing ets proteins from a variety of species has been developed. This antibody recognizes ets1, ets2, erg, and other related proteins. It has a high affinity for the ets1 protein. The epitope for the pan ets mAb consists of about 13 amino acids. This antibody can be used to isolate and characterize new members of ets gene family derived from a c-DNA expression library, as well as to identify other "ets motif" binding proteins.

Amino Acid Sequence↗
PubMed1991

IL-1 induces ornithine decarboxylase in normal T lymphocytes.

IL-1 alpha regulation of ornithine decarboxylase (ODC) was examined in T cells because IL-1 is a costimulus for T cell proliferation and ODC is a critical enzyme in the metabolic events associated with cellular proliferation. In the present study, we demonstrate that IL-1 alpha induces ODC mRNA and ODC enzyme activity in T cells. Unlike many IL-1 actions on T cells, this did not require a costimulus from the TCR, IL-1 alone being sufficient to induce ODC. The mechanism of IL-1 induction of ODC probably operates at several levels, including transcription, mRNA stability, and translation. Previous studies have shown that IL-1 prepares T cells for replication by increasing the production of c-jun, c-fos, c-myc, growth factors, growth factor receptors, and the response to growth factors. From the present study, ODC induction can be added to the list of IL-1-induced replicative machinery in T cells.

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

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

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

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

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↗