Biomedical subjects

T Shenk

Publications and source records attributed to T Shenk.

At least 55 records · Page 3Linked to original sources

PubMed1996

Protection against apoptosis by the vaccinia virus SPI-2 (B13R) gene product.

Vaccinia virus contains a gene, termed SPI-2 or B13R, that is closely related in its sequence to a potent inhibitor of apoptosis from cowpox virus (crmA). Infection by vaccinia virus protects HeLa cells against apoptosis that is induced by an immunoglobulin M antibody against the fas receptor or by tumor necrosis factor alpha. This effect is profoundly reduced when the SPI-2 gene is deleted. The SPI-2 gene, when transiently expressed in these cells, can also protect against apoptosis mediated by these agents. Given the similarity to crmA, it seems likely that SPI-2 functions in an analogous fashion, inhibiting the activity of ICE protease family members and blocking the onset of apoptosis.

Apoptosis↗

PubMed1996

The MDM2 oncoprotein binds specifically to RNA through its RING finger domain.

BACKGROUND: The cellular mdm2 gene has transforming activity when overexpressed and is amplified in a variety of human tumors. At least part of the transforming ability of the MDM2 protein is due to binding and inactivating the p53 tumor suppressor protein. Additionally, this protein forms a complex in vivo with the L5 ribosomal protein and its associated 5S ribosomal RNA and may be part of a ribosomal complex. MATERIALS AND METHODS: A RNA homopolymer binding assay and a SELEX procedure have been used to characterize the RNA-binding activity of MDM2. RESULTS: The MDM2 protein binds efficiently to the homopolyribonucleotide poly(G) but not to other homopolyribonucleotides. This binding is independent of the interaction of MDM2 with the L5 protein, which occurs through the central acidic domain of MDM2. An RNA SELEX procedure was performed to identify specific RNA ligands that bind with high affinity to the human MDM2 (HDM2) protein. After 10 rounds of selection and amplification, a subset of RNA molecules that bound efficiently to HDM2 was isolated from a randomized pool. Sequencing of these selected ligands revealed that a small number of sequence motifs were selected. The specific RNA binding occurs through the RING finger domain of the protein. Furthermore, a single amino acid substitution in the RING finger domain, G446S, completely abolishes the specific RNA binding. CONCLUSIONS: These observations, showing that MDM2 binds the L5/5S ribosomal ribonucleoprotein particle and can also bind to specific RNA sequences or structures, suggest a role for MDM2 in translational regulation in a cell.

Amino Acid Sequence↗

PubMed1995

DNA binding sites for the transcriptional activator/repressor YY1.

YY1 is ubiquitously expressed zinc finger DNA binding protein. It can act as a transcriptional repressor or activator and, when binding at the initiator element, as a component of the basal transcription complex. Binding sites for YY1 have been reported in a wide variety of promoters and they exhibit substantial diversity in their sequence. To better understand how YY1 interacts with DNA and to be able to predict the presence of YY1 sites in a more comprehensive fashion, we have selected YY1 binding sites from a random pool of oligonucleotides. The sites display considerable heterogeneity, but contain a conserved 5'-CAT-3' core flanked by variable regions, generating the consensus 5'-(C/g/a)(G/t)(C/t/a)CATN(T/a)(T/g/c)-3', where the upper case letters represent the preferred base. This high degree of flexibility in DNA recognition can be predicted by modeling the interaction of the four YY1 zinc fingers with DNA and a detailed model for this interaction is presented and discussed.

Amino Acid Sequence↗

PubMed1995

Differentiation is induced in three-dimensional cultures of brain cells immortalized by the LAP mammalian regulatory system.

Immortalized neuroectodermal precursor cell lines were generated from mouse brain by the SV40 large T antigen expressed under the control of the LAP (lac activating protein) mammalian regulatory system. The LAP system permits the reversible expression of T antigen as a function of the exogenous inducer, isopropyl-beta-D-thiogalactopyranoside. Immortalized cells can be stably maintained in an undifferentiated state in monolayer cultures. Cell lines expressed the early neurofilament-like protein nestin, but not markers characteristic for mature cells such as the neurofilament light protein and glial fibrillary acidic protein. Downregulating the LAP-controlled T antigen with isopropyl-beta-D-thiogalactopyranoside was not sufficient to induce differentiation. However, when cells formed three-dimensional aggregates, differentiation to a neuronal phenotype occurred, indicating that cell-cell interaction plays an important role in their differentiation. Cells in aggregates did not proliferate, even in the presence of T antigen, suggesting that an aggregation-induced signal to cease growth was dominant over the growth signal of T antigen. Further morphological differentiation was induced by basic fibroblast growth factor. These immortalized cells should facilitate molecular and cellular studies concerned with the mechanism of commitment, fate determination, and mitotic arrest of neuronal precursor cells in the developing mammalian CNS.

Animals↗

PubMed1995

Human cytomegalovirus IE1 and IE2 proteins block apoptosis.

Human cytomegalovirus-infected fibroblasts are resistant to the induction of apoptosis by superinfection with a mutant adenovirus unable to produce the viral E1B 19-kDa protein that normally causes an E1A protein-mediated apoptotic response. Two cytomegalovirus gene products that block apoptosis were identified. The IE1 and IE2 proteins each inhibit the induction of apoptosis by tumor necrosis factor alpha or by the E1B 19-kDa-protein-deficient adenovirus but not by irradiation with UV light. Our results suggest a new physiological role for the IE1 and IE2 proteins in the human cytomegalovirus replication cycle.

Animals↗

PubMed1995

In vitro selection of RNA ligands for the ribosomal L22 protein associated with Epstein-Barr virus-expressed RNA by using randomized and cDNA-derived RNA libraries.

The Epstein-Barr virus (EBV)-expressed RNA 1 (EBER1) associates tightly with the ribosomal protein L22. We determined the general requirements for an RNA to bind L22 in a SELEX experiment, selecting RNA ligands for L22 from a randomized pool of RNA sequences by using an L22-glutathione S-transferase fusion protein. The selected sequences all contained a stem-loop motif similar to that of the region of EBER1 previously shown to interact with L22. The nucleotides were highly conserved at three positions within the stem-loop and identical to the corresponding nucleotides in EBER1. Two independent binding sites for L22 could be identified in EBER1, and mobility shift assays indicated that two L22 molecules can interact with EBER1 simultaneously. To search for a cellular L22 ligand, we constructed a SELEX library from cDNA fragments derived from RNA that was coimmunoprecipitated with L22 from an EBV-negative whole-cell lysate. After four rounds of selection and amplification, most of the clones that were obtained overlapped a sequence corresponding to the stem-loop between nucleotides 302 and 317 in human 28S ribosomal RNA. This stem-loop fulfills the criteria for optimal binding to L22 that were defined by SELEX, suggesting that human 28S ribosomal RNA is likely to be a cellular L22 ligand. Additional L22 binding sites were found in 28S ribosomal RNA, as well as within 18S ribosomal RNA and in RNA segments not present in sequence databases. The methodology described for the conversion of a preselected cellular RNA pool into a SELEX library might be generally applicable to other proteins for the identification of cellular RNA ligands.

Base Sequence↗

PubMed1995

Adenovirus E1A proteins interact with the cellular YY1 transcription factor.

The adenovirus 12S and 13S E1A proteins have been shown to relieve repression mediated by the cellular transcription factor YY1. The 13S E1A protein not only relieves repression but also activates transcription through YY1 binding sites. In this study, using a variety of in vivo and in vitro assays, we demonstrate that both E1A proteins can bind to YY1, although the 13S E1A protein binds more efficiently than the 12S E1A protein. Two domains on the E1A proteins interact with YY1: an amino-terminal sequence (residues 15 to 35) that is present in both E1A proteins and a domain that includes at least a portion of conserved region 3 (residues 140 to 188) that is present in the 13S but not the 12S E1A protein. Two domains on YY1 interact with E1A proteins: one is contained within residues 54 to 260, and the other is contained within the carboxy-terminal domain of YY1 (residues 332 to 414). Cotransfection of a plasmid expressing carboxy-terminal amino acids 332 to 414 of YY1 fused to the GAL4 DNA-binding domain can inhibit expression from a reporter construct with GAL4 DNA binding sites in its promoter, and inclusion of a third plasmid expressing E1A proteins can relieve the repression. Thus, we find a correlation between the ability of E1A to interact with the carboxy-terminal domain of YY1 and its ability to relieve repression caused by the carboxy-terminal domain of YY1. We propose that E1A proteins normally relieve YY1-mediated transcriptional repression by binding directly to the cellular transcription factor.

Adenovirus E1A Proteins↗

PubMed1995

Two domains of p53 interact with the TATA-binding protein, and the adenovirus 13S E1A protein disrupts the association, relieving p53-mediated transcriptional repression.

The tumor suppressor gene product p53 can activate and repress transcription. Both transcriptional activation and repression are thought to involve the direct interaction of p53 with the basal transcriptional machinery. Previous work has demonstrated an in vitro interaction between p53 and the TATA-binding protein that requires amino acids 20 to 57 of p53 and amino acids 220 to 271 of the TATA-binding protein. The present results show that a 75-amino-acid segment from the carboxy terminus of p53 also can bind to the TATA-binding protein in vitro, and this interaction requires amino acids 217 to 268 of the TATA-binding protein, essentially the same domain that is required for interaction with the amino-terminal domain of p53. A carboxy-terminal segment of p53 can mediate repression when bound to DNA as a GAL4-p53 fusion protein. The amino- and carboxy-terminal p53 interactions occur within the domain on the TATA-binding protein to which the adenovirus 13S E1A oncoprotein has previously been shown to bind. The 13S E1A oncoprotein can dissociate the complex formed between the carboxy-terminal domain of p53 and the TATA-binding protein and relieve p53-mediated transcriptional repression. These results demonstrate that two independent domains of p53 can potentially interact with the TATA-binding protein, and they define a mechanism--relief of repression--by which the 13S E1A oncoprotein can activate transcription through the TATA motif.

Adenovirus E1A Proteins↗

PubMed1994

Relief of p53-mediated transcriptional repression by the adenovirus E1B 19-kDa protein or the cellular Bcl-2 protein.

The p53 tumor suppressor gene product is a transcriptional regulatory protein. It activates transcription from promoters that contain a p53 DNA binding site but represses many promoters that lack its binding site. High-level expression of wild-type p53 can induce apoptosis in certain cell types, and this activity can be blocked by the adenovirus E1B 19-kDa oncoprotein or by the cellular Bcl-2 oncoprotein. Here we report that p53-mediated repression of promoters that lack a p53 binding site is abrogated by the E1B 19-kDa protein or Bcl-2 oncoprotein. In contrast, transcriptional activation by p53 still occurs in the presence of either protein. The fact that two oncoproteins capable of preventing p53-mediated apoptosis also block transcriptional repression by p53 raises the possibility that p53 might induce apoptosis, at least in part, by repressing transcription.

Adenovirus E1B Proteins↗

PubMed1994

TATA-binding protein-independent initiation: YY1, TFIIB, and RNA polymerase II direct basal transcription on supercoiled template DNA.

YY1 is a zinc finger transcription factor whose DNA-binding motif exhibits the properties of an initiator element. Only three factors were required to direct specific basal transcription on a supercoiled template DNA carrying the YY1 initiator: YY1, general transcription factor IIB, and RNA polymerase II. This minimal in vitro reaction did not require the TATA-binding protein (TBP). We propose that, under appropriate conditions, YY1 can function like TBP, as a factor that binds to the core promoter and recruits the polymerase to the initiation complex.

Adenoviridae↗

PubMed1994

The 5' flanking region of the serotonin 2 receptor gene directs brain specific expression in transgenic animals.

The neuron is the predominant cell type expressing the serotonin 2 (5-HT2) receptor in the central nervous system. Transcriptional control elements involved in the restriction of 5-HT2 receptor gene expression to neuronal cells and tissues were studied using both transgenic mice and cultured cells. Sequences extending from a site near the translational initiation codon to -5.6 kb in the 5' flanking region of the murine receptor gene were found to be sufficient to target gene expression to the brain in transgenic animals. In transient transfection experiments a basal promoter was identified which was functional in both neuronal and nonneuronal cells. Upstream of the basal promoter two repressor domains were found within the 5' flanking sequence of the receptor gene. These sequences repressed gene activity in all cells except cells of neuronal origin, thus the repressor domains are the primary determinants to generate neuronal cell-specific transcription of the 5-HT2 receptor gene.

Animals↗

PubMed1994

The 64-kilodalton subunit of the CstF polyadenylation factor binds to pre-mRNAs downstream of the cleavage site and influences cleavage site location.

The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H-mediated mapping technique, we show that the 64-kDa subunit of CstF can be photo cross-linked to pre-mRNAs at U-rich regions located downstream of the cleavage site of the simian virus 40 late and adenovirus L3 pre-mRNAs. This positional specificity of cross-linking is a consequence of CstF interaction with the polyadenylation complex, since the 64-kDa protein by itself is cross-linked at multiple positions on a pre-mRNA template. During polyadenylation, four consecutive U residues can substitute for the native downstream U-rich sequence on the simian virus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch not only enables the 64-kDa polypeptide to be cross-linked to the pre-mRNA but also influences the site of cleavage. A search of the GenBank database revealed that a substantial portion of mammalian polyadenylation sites carried four or more consecutive U residues positioned so that they should function as sites for interaction with the 64-kDa protein downstream of the cleavage site. Our results indicate that the polyadenylation machinery physically spans the cleavage site, directing cleavage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa subunit of CstF.

Adenoviridae↗

PubMed1994

Antagonist-mediated down-regulation of 5-hydroxytryptamine type 2 receptor gene expression: modulation of transcription.

Prolonged exposure to an agonist results in a progressive loss of most G protein-coupled receptors, whereas exposure to an antagonist causes increased receptor response. The 5-hydroxytryptamine (5-HT)2 receptor is down-regulated by agonists but, paradoxically, antagonists can also elicit a decrease in receptor density. Here we show that long term treatment with serotonin or mianserin, an antagonist and antidepressant, results in reduced levels of both the 5-HT2 receptor and its RNA. Antagonist-induced down-regulation requires the presence of the 5-HT2 receptor, it occurs at the level of transcription, and it is mediated by a drug response sequence in the 5' flanking region of the 5-HT2 receptor gene. The effect of mianserin might result, at least in part, from its ability to modulate transcription.

Animals↗

PubMed1994

Adenovirus E1A expression controlled by the red pigment promoter in transgenic mice: a model for developmental abnormalities of the eye.

Four transgenic founder mice were produced that carry the adenovirus E1A gene under control of the human red pigment promoter. Two of the founder animals passed the transgene to progeny, and one line was bred to homozygosity. The line of mice homozygous for the transgene expressed E1A-specific RNA at a low level in their eyes; no expression could be detected in other tissues that were tested. Either the founder animals or their progeny exhibited developmental abnormalities of their eyes, including a small eye phenotype, retinal dysplasia, anterior cleavage syndrome, and retinal pigment epithelium dysplasia. Individuals from the same line of mice exhibited subsets of the abnormalities, e.g., many animals had one normal size eye and one small eye, even though both eyes contained E1A-specific RNA. No tumors resulted from E1A expression in animals monitored for 22 months.

Adenovirus E1A Proteins↗

PubMed1993

Interaction between transcription factors Sp1 and YY1.

A basal level of transcription is usually observed when all but a small region of DNA has been deleted from a eukaryotic gene promoter. These promoter elements, which are necessary and sufficient for specific transcription initiation, are referred to as minimal or core promoter elements. One element that is commonly present in a core promoter is the initiator. It has been demonstrated that the presence of Sp1 binding sites can greatly enhance the level of transcription initiation at initiator elements. A binding site for the YY1 transcription factor, located at the initiation site of the adeno-associated virus P5 promoter, functions as an initiator element; a synergistic enhancement of its activity is observed in vitro when upstream Sp1 binding sites are present. Here we report that this synergistic activation probably occurs through protein-protein interactions.

Animals↗

PubMed1993

Glial cell-specific expression of the serotonin 2 receptor gene: selective reactivation of a repressed promoter.

The 5' flanking region of the 5-HT2 receptor gene has been cloned, sequenced and its transcriptional regulatory functions analyzed. The promoter lacks an identifiable TATA motif, and utilizes at least 11 clustered start sites. Promoter function was analyzed by transient assays in rat C6 glioma cells, which were shown to express the endogenous 5-HT2 receptor gene, as well as in rat CREF and human HeLa cells which do not express the endogenous gene. The basal promoter functioned equally well in all three cell lines; and a repression domain, located upstream of the basal promoter, inhibited activity of the promoter in all three cell lines. A far upstream cell specific activator domain restored promoter activity in C6 glioma cells, but did not reactivate the silenced promoter in CREF or HeLa cells. The upstream activator domain, repressor domain and basal promoter functioned in concert to achieve cell type specific expression. The activator domain did not direct C6 glioma cell specific expression in the absence of the repressor domain or in constructs carrying a heterologous basal promoter. These results indicate that glial cell expression of the 5-HT2 receptor gene is achieved through a cell type specific reactivation of a repressed promoter.

Amino Acid Sequence↗

PubMed1993

Adenovirus E4orf4 protein binds to protein phosphatase 2A, and the complex down regulates E1A-enhanced junB transcription.

Adenovirus E4orf4 protein was previously shown to counteract transactivation of junB by cyclic AMP (cAMP) and E1A protein. It was also shown to cause hypophosphorylation of E1A and c-Fos proteins. Here we show that the E4orf4 protein associates with protein phosphatase 2A. All three subunits of the phosphatase are present in the complex, and the B subunit interacts directly with the viral protein. The complex possesses a phosphatase activity typical of protein phosphatase 2A, and the phosphatase mediates the E4orf4-induced down regulation of junB transcription. Thus, adenovirus E4orf4 protein recruits protein phosphatase 2A into a signal transduction pathway initiated by cAMP and E1A protein.

Adenovirus E1A Proteins↗

PubMed1993

The adenovirus L3 23-kilodalton proteinase cleaves the amino-terminal head domain from cytokeratin 18 and disrupts the cytokeratin network of HeLa cells.

Immunofluorescence studies revealed that adenovirus induces a reorganization of the cytokeratin system in lytically infected HeLa cells. At 24 h postinfection, the cytokeratin network began to disassemble into prominent spheroid globules. By 36 h postinfection, host cell lysis occurred, accompanied by the formation of perinuclear cytokeratin clumps and additional spheroid globules. Immunoblots detected 41- and 44-kDa fragments of cytokeratin 18 and reduced levels of cytokeratin 7 at 24 and 36 h postinfection. Cytokeratin proteins isolated from HeLa cells at 36 h postinfection were deficient in filament polymerization. The 41-kDa proteolytic cytokeratin 18-specific fragment was purified, and its amino-terminal sequence was determined to be GGIQNEKETM. These residues correspond to amino acids 74 through 83 of cytokeratin 18, identifying a cleavage site at the junction of the globular head domain and the alpha-helical rod domain. Moreover, this truncation event occurs at a consensus cleavage site for the adenovirus L3 23-kDa proteinase. The temperature-sensitive mutant H2-ts1, which contains a mutation in the proteinase, neither induced cleavage of cytokeratin 18 nor precipitated the formation of spheroid globules during lytic infection at the nonpermissive temperature. The active proteinase is therefore required for cleavage of cytokeratin 18 and morphological rearrangement of the cytokeratins. We suggest that disruptions in the cytokeratin system weaken the mechanical integrity of the cell, thus promoting host cell lysis and release of progeny virions.

Adenoviruses, Human↗