Biomedical subjects

T Subramanian

Publications and source records attributed to T Subramanian.

At least 55 records · Page 3Linked to original sources

PubMed1993

Mutational analysis of the transforming and apoptosis suppression activities of the adenovirus E1B 175R protein.

The role of the adenovirus-2 E1B 19-kDa (175R) T antigen in E1a-cooperative transformation was determined by cotransfection of plasmids expressing E1A or E1B 175R T antigens into primary rat kidney (BRK) cells. Transformed cells were selected by virtue of their resistance to the antibiotic Geneticin (G418) conferred by neo gene co-expression from plasmids coding for 175R. 175R cooperated efficiently with genomic E1a and specifically with the 289R protein coded by the 13S mRNA in the transformation of primary BRK cells. Mutational analysis of the 175R protein revealed that the N terminus and the C-terminal 30 amino acids are not essential for E1a-cooperative transformation. Several conserved sequences located in the middle of the 175R protein are essential for transformation. The effect of various mutants to suppress apoptosis (programmed cell death) induced by an anti-cancer agent, cisplatin, was examined in cells producing the E1A and E1B 175R proteins. Apoptosis was measured by flow cytometric analysis and indicates that the 175R protein efficiently prevents cisplatin-induced apoptosis. This suggests that the 175R function involved in transformation segregates with its ability to suppress cisplatin-induced apoptosis.

Adenovirus E1A Proteins↗

PubMed1993

A region in the C-terminus of adenovirus 2/5 E1a protein is required for association with a cellular phosphoprotein and important for the negative modulation of T24-ras mediated transformation, tumorigenesis and metastasis.

We have examined a series of small deletion mutants within exon 2 of the adenovirus 2/5 E1A oncogene product, the 243R protein, for immortalization, ras cooperative transformation, tumorigenesis and metastasis. Compared with wild-type 243R, various deletion mutants located between residues 193 and 243 cooperated more efficiently with ras to induce large transformed foci of less adherent cells that were tumorigenic and metastatic. However, the greatest enhancement of transformation (comparable to that obtained with a deletion of the C-terminal 67 amino acids) was observed with a mutant carrying a deletion of residues 225-238. This mutant was also more defective in immortalization. These results suggest that this 14 amino acid region may contain a function that is important for immortalization and negative modulation of tumorigenesis and metastasis. To identify cellular proteins that may associate with the exon 2-coded region of E1A (C-terminal half) and modulate its transformation potential, we constructed a chimeric gene coding for the C-terminal 68 amino acids of E1a fused to bacterial glutathione-S-transferase (GST). This fusion protein was used to purify cellular proteins that bind to the C-terminal region of E1a. A 48 kDa cellular protein doublet (designated CtBP) was found to bind specifically to the GST-E1a C-terminal fusion protein as well as to bacterially expressed full-length E1a (243R) protein. It also co-immunoprecipitated specifically with E1a. Analysis of a panel of GST-E1a C-terminal mutant proteins indicates that residues 225-238 are required for the association of E1a and CtBP, suggesting a correlation between the association of CtBP and the immortalization and transformation modulating activities of exon 2. CtBP is a phosphoprotein and the level of phosphorylation of CtBP appears to be regulated during the cell cycle, suggesting that it may play an important role during cellular proliferation.

Adenovirus E1A Proteins↗

PubMed1993

Activation of a heterologous promoter by human immunodeficiency virus type 1 Tat requires Sp1 and is distinct from the mode of activation by acidic transcriptional activators.

We have previously shown that the Tat protein of the human immunodeficiency virus type 1 (HIV-1) is a modular transcriptional activator that can be targeted upstream of either a synthetic promoter or the intact HIV promoter to activate transcription. This activation was shown to be largely dependent on the presence of consensus binding sites for the cellular transcription factor Sp1. Since the use of heterologous promoters may provide further insight into Tat-mediated transactivation, we have analyzed the transactivation of the thymidine kinase promoter of herpes simplex virus by Tat and by the acidic transcriptional transactivator VP16. The effects of mutations of defined upstream promoter elements show that Tat transactivation is dependent on Sp1 binding sites in a site-specific manner. In contrast, transactivation by the acidic transactivator VP16 is completely independent of any of the defined promoter elements upstream of the TATA box. These results suggest that Tat and the classically defined modular acidic transcriptional activators have different modes of transactivation. In addition, the substitution of the HIV-1 TATA box for the thymidine kinase TATA box substantially increases Tat transactivation, indicating that Tat transactivation may also ultimately involve TATA box-associated cellular transcription factors.

Base Sequence↗

PubMed1992

A high-level expression vector for human cells.

We have developed a vector (pSupexp), for high-level expression of genes, that is dependent on transactivation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) by the HIV-1 transactivator protein, Tat. The foreign gene, expressed under transcriptional control of the HIV-1 LTR, and the tat gene, expressed under transcriptional control of SV40 early promoter, are expressed from the same plasmid. The vector also has the neomycin resistance-encoding gene (neo), with G418 being used as a dominant selection marker for stable expression. We have cloned the bacterial cat gene into pSupexp and measured transient CAT production in human HeLa and A549 cells. Our results indicate that pSupexpCAT expresses about 25- to 68-fold higher levels of CAT activity as compared to other standard SV40- and Rous sarcoma virus-based vectors, and three- to fivefold more activity than the cytomegalovirus-based vector. Immunoprecipitation of the CAT protein also revealed a high level of production in human cells.

Cell Line↗

PubMed1992

Functional comparison of the basic domains of the Tat proteins of human immunodeficiency virus types 1 and 2 in trans activation.

The trans-activator Tat proteins coded by human immunodeficiency virus type 1 (HIV-1) and HIV-2 appear to be similar in structure and function. However, the Tat protein of HIV-2 (Tat2) activates the HIV-1 long terminal repeat (LTR) less efficiently than Tat1 (M. Emerman, M. Guyader, L. Montagnier, D. Baltimore, and M. A. Muesing, EMBO J. 6:3755-3760, 1987). To determine the functional domain of Tat2 which contributes to this incomplete reciprocity, we have carried out domain substitution between Tat1 and Tat2 by exchanging the basic domains involved in Tat interaction with its target trans-activation-response (TAR) RNA structure. Our results indicate that Tat1 proteins containing substitutions of either 8 or 14 amino acids of the basic domain of Tat2 exhibited reduced trans activation of the HIV-1 LTR by about 1/20 or one-fourth the level induced by wt Tat1. In contrast, Tat2 containing a substitution of the 9-amino-acid basic domain of Tat1 trans activated HIV-1 LTR like native Tat1. A substitution of the highly conserved core domain of Tat2 with that of Tat1 did not have any significant effect on trans activation of the HIV-1 LTR. These results indicate that the basic domain of Tat2 contributes to its inefficient trans activation of the HIV-1 LTR. Mutation of an acidic residue (Glu) located between the core domain and the Arg-rich basic domain of Tat2 at position 77 to a Gly residue increased the activity of Tat2 substantially. These results further suggest that the presence of an acidic residue (Glu) adjacent to Arg-rich sequences may at least partially contribute to the reduced activity of the Tat2 basic domain.

Amino Acid Sequence↗

PubMed1991

Sp1-dependent activation of a synthetic promoter by human immunodeficiency virus type 1 Tat protein.

The Tat protein coded by human immunodeficiency virus (HIV) is a strong activator of viral gene expression from the long terminal repeat (LTR). It appears that Tat-mediated trans-activation of the HIV LTR is predominantly a transcriptional event. It has been reported that Tat acts at the level of both transcriptional initiation and elongation through interaction with a nascent RNA target sequence termed TAR (for trans-activation response element). However, the precise mechanism(s) by which Tat mediates TAR-dependent transcriptional activity is not known. To determine whether Tat functions similarly to other eukaryotic transcriptional activators through any of the conventional promoter elements, we tested Tat activity on synthetic promoters containing consensus sequences required for binding transcription factor Sp1 and a TATA box. Here, we report that a chimeric Tat protein targeted to the promoter region by the DNA-binding domain of yeast transcription factor GAL4 activates the synthetic promoter. Because this trans-activation depends on Sp1-binding sites, Tat can apparently mediate transcriptional activation through its interaction with Sp1. Mutational analysis of the gal4-tat chimeric gene reveals that the N-terminal 48-amino acid region of Tat constitutes the activation region for Sp1-dependent trans-activation. This region of Tat exhibits substantially more activity than the N-terminal 58 amino acids of Tat, which includes the arginine-rich basic region. Effects of specific mutations in the 48-amino acid Tat region of GAL4-Tat on trans-activation of the synthetic promoter mimic the effects of these specific mutations on Tat-mediated trans-activation of the HIV-1 LTR, suggesting that trans-activation of both the synthetic promoter and the intact LTR occurs by a common mechanism.

Base Sequence↗

PubMed1991

Heterologous basic domain substitutions in the HIV-1 Tat protein reveal an arginine-rich motif required for transactivation.

The Tat protein coded by HIV-1 is a unique eukaryotic transactivator. It activates gene expression from the viral LTR by its interaction with a nascent RNA element (TAR) located at the 5' end of all HIV-1 transcripts. Tat appears to bind to its target RNA structure in a highly sequence-specific manner. The TAR-binding activity of Tat has been localized in an Arg-rich basic domain located between residues 49 and 57 of the Tat protein. We have carried out domain substitution studies with heterologous basic domains which are also implicated in RNA binding. Here, we report that a 19 or a 12 amino acid region from the N-terminus of HTLV-I Rex can functionally substitute for the Tat basic domain. In contrast, the Arg-rich domains of the N gene products of bacteriophages lambda and 21 do not functionally substitute for the Tat basic domain. The positive and negative effects of various domain substitution mutants have facilitated identification of a consensus sequence (Arg/Lys-X-X-Arg-Arg-X-Arg-Arg) in the basic domain required for Tat activity. Conversion of the functionally inactive basic domain of the lambda N protein to the consensus motif restored the transactivation function of the Tat-N chimeric protein. Similarly, the Rex basic domain containing scrambled sequences unrelated or partially related to the consensus motif were either totally defective in transactivation or exhibited reduced activity. Our results further suggest that the activity of the core Arg motif may be enhanced by the presence of Gln or Asn within the basic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Requirement of the C-terminal region of adenovirus E1a for cell transformation in cooperation with E1b.

We have previously reported that adenovirus E1a mutants lacking the C-terminal 61 or 67 amino acids were severely defective in immortalization, but cooperated more efficiently (than wt E1a) with activated T24 ras oncogene in transformation of primary rat kidney (BRK) cells (Subramanian et al., 1989; Oncogene, 4:415-420). Here, we show that in contrast to these previous results, transformation of BRK cells in cooperation with the Ad2 E1b region is dependent on the C-terminal region of E1a. Mutational analysis of the C-terminal region has revealed that a region located between residues 266 and 276 may be important for E1a/E1b cooperative transformation. Like E1a/T24 ras cooperative transformation, E1a/E1b cooperative transformation also requires two essential domains involved in the binding of two cellular proteins (300K and 105K) within the N-terminal half of E1a. E1a/E1b cooperative transformation therefore requires an additional E1a activity encoded within the C-terminal region of E1a.

Adenoviridae↗

PubMed1990

Functional substitution of the basic domain of the HIV-1 trans-activator, Tat, with the basic domain of the functionally heterologous Rev.

The tat gene of HIV is a strong activator of the viral LTR. The Tat protein contains a highly basic domain that is important for its transport to the nuclear/nucleolar locations. The Tat basic domain when fused to Escherichia coli beta-galactosidase directed the chimeric protein to the nucleus and nucleolus. Tat mutants lacking the entire basic domain were severely defective in trans-activation. Substitution of the basic domain of Tat with that of the functionally unrelated HIV-1 Rev protein targeted the chimeric protein to the nucleolus and restored the function of Tat. In contrast, substitution with the nuclear targeting signal (NLS) of SV40 T antigen targeted the chimeric protein to the nucleus and accumulation in the nucleolar region was excluded. The Tat-NLS chimeric protein did not restore the trans-activation function of Tat efficiently. These results indicate that the arginine-rich basic domain of the trans-activator, Tat, and post-transcriptional trans-regulator, Rev, are functionally similar with regard to trans-activation of HIV-1 LTR.

Amino Acid Sequence↗

PubMed1990

Selective induction of toxicity to human cells expressing human immunodeficiency virus type 1 Tat by a conditionally cytotoxic adenovirus vector.

The human immunodeficiency viruses (HIVs) primarily infect CD4+ T lymphocytes, leading eventually to the development of a systemic immune dysfunction termed acquired immunodeficiency syndrome (AIDS). An attractive strategy to combat HIV-mediated pathogenesis would be to eliminate the initial pool of infected cells and thus prevent disease progression. We have engineered a replication-defective, conditionally cytotoxic adenovirus vector, Ad-tk, whose action is dependent on the targeted expression of the herpes simplex virus type 1 thymidine kinase gene (tk), cloned downstream of the HIV-1 long terminal repeat, in human cells expressing the HIV-1 transcriptional activator Tat. Infection of Tat-expressing human HeLa or Jurkat cells with Ad-tk resulted in high-level tk expression, which was not deleterious to the viability of these cells. However, in the presence of the antiherpetic nucleoside analog ganciclovir, Ad-tk infection resulted in a massive reduction in the viability of these Tat-expressing cell lines. As adenoviruses are natural passengers of the human lymphoid system, our results suggest adenovirus vector-based strategies for the targeted expression, under the control of cis-responsive HIV regulatory elements, of cytotoxic agents in HIV-infected cells for the therapy of HIV-mediated pathogenesis.

Adenoviruses, Human↗

PubMed1990

Effects of placental dressing indolent ulcers.

Fifteen cases of indolent ulcers of varying aetiology were treated by human placental dressing. An equal number of patients treated with antibiotics were taken as control. Human placental dressing for chronic ulcers was found to be effective in wound healing, inexpensive, freely available and devoid of side-effects. The immunological response which produces the clinical results by the use of human placenta has been studied. The raised level of immunoglobulins in the form of IgG and IgM was also seen.

Adolescent↗

PubMed1989

Multiple functional domains of Tat, the trans-activator of HIV-1, defined by mutational analysis.

The tat gene of HIV-1 is a potent trans-activator of gene expression from the HIV long terminal repeat (LTR). To define the functionally important regions of the product of the tat gene (Tat) of HIV-1, deletion, linker insertion and single amino acid substitution mutants within the Tat coding region of strain SF2 were constructed. The effect of these mutations on trans-activation was assessed by measuring the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene linked to the HIV-LTR. These studies have revealed that four different domains of the protein that map within the N-terminal 56 amino acid region are essential for Tat function. In addition to the essential domains, an auxiliary domain that enhances the activity of the essential region has also been mapped between amino acid residues 58 and 66. One of the essential domains maps in the N-terminal 20 amino acid region. The other three essential domains are highly conserved among the various strains of HIV-1 and HIV-2 as well as simian immunodeficiency virus (SIV). Of the conserved domains, one contains seven Cys residues and single amino acid substitutions for several Cys residues indicate that they are essential for Tat function. The second conserved domain contains a Lys X Leu Gly Ile X Tyr motif in which the Lys residue is essential for trans-activation and the other residues are partially essential. The third conserved domain is strongly basic and appears to play a dual role. Mutants lacking this domain are deficient in trans-activation and in efficient targeting of Tat to the nucleus and nucleolus. The combination of the four essential domains and the auxiliary domain contribute to the near full activity observed with the 101 amino acid Tat protein.

Amino Acid Sequence↗

PubMed1989

Enhanced ras oncogene mediated cell transformation and tumorigenesis by adenovirus 2 mutants lacking the C-terminal region of E1a protein.

Mutants of adenovirus 2 E1a defective in coding for the C-terminal 61 or 67 amino acids of a 243 amino acid (243R) protein are defective in immortalization of primary baby rat kidney (BRK) cells. However, they cooperate with T24 ras in oncogenic transformation more efficiently than wt. BRK cells transformed by the E1a C-terminal mutants and T24 ras induce rapidly growing tumors in syngeneic rats and athymic mice whereas cells transformed by the wt 243R and ras oncogene are not tumorigenic in syngeneic rats and can only induce slowly growing tumors in athymic mice. Cells transformed by the E1a mutants and ras oncogene also induce rapid metastatic tumors whereas cells transformed by the wt 243R and T24 ras can not do so. The increased tumorigenic ability exhibited by the 243R mutants does not appear to be due to differential levels of expression of p21 ras. Our results suggest that the C-terminal region of the 243R protein may have a novel function in suppression of cell transformation, tumorigenesis and tumor progression.

Adenoviridae↗

PubMed1988

An N-terminal region of adenovirus E1a essential for cell transformation and induction of an epithelial cell growth factor.

A new region of the adenovirus E1a protein essential for immortalization and transformation of primary rat kidney cells has been identified. This region is located between amino acid residues 18 to 20 in an N-terminal domain that is not conserved among the various adenovirus serotypes. The transformation defective mutant (18-0) mapping in this region is not impaired in its ability to trans-activate the viral E2 promoter and to repress the activity of certain enhancer elements. Mutant 18-0 appears to have only a partial defect in the induction of cellular DNA synthesis in quiescent primary cells suggesting that the N-terminal region plays a role in immortalization and transformation by a mechanism that may not fully depend on induction of cellular DNA synthesis. Mutant 18-0 and another transformation defective mutant (125-7) mapping between amino acid residues 125 to 127 in a conserved domain are defective in the induction of an epithelial cell growth factor, suggesting that growth factor induction may be important for some aspect of adenovirus mediated immortalization and transformation.

Adenovirus Early Proteins↗

PubMed1988

Separation of immortalization and T24-ras oncogene cooperative functions of adenovirus E1a.

An adenovirus 2 E1a gene coding for a protein of 243 (243R) amino acids can efficiently immortalize primary rat kidney (BRK) cells and cooperate with the activated cellular ras oncogene (T24 ras). A mutant (47-0) of the 243R gene that maps between amino acid residues 47-50 within a region that is highly conserved among the various adenovirus serotypes was found to be severely defective in immortalization. Despite the defect in immortalization, mutant 47-0 had the ability to cooperate with T24 ras in oncogenic transformation. These results suggest that the immortalization and the oncogene cooperation functions of the 243R are separable. Our results further suggest that the requirement for a separate immortalization function can be circumvented by oncogenic transformation and that the immortalization of cells transformed by E1a and T24 ras may be a secondary consequence of transformation by these two oncogenes.

Adenovirus Early Proteins↗

PubMed1986

Separation of the functions controlled by adenovirus 2 lp+ locus.

The adenovirus lp+ locus is located within early region E1b (map position 4.5-11.2) and codes for a 19-kDa tumor antigen (175R). Genetic analysis of the viral mutants that map within this region indicates that the lp+ locus controls multiple functions in cell transformation and in productive viral infection. Viral mutants mapping within the lp+ locus produce wt-like cytopathic effect (cyt+) or a cytocidal (cyt) effect. Earlier results have shown that many of the viral mutants that produce cyt phenotype in infected cells are transformation defective. In the present studies we show that one of the cyt mutants, cyt 106 which has a single amino acid substitution at position 20 transforms the established rat embryo cell line, CREF, at somewhat reduced frequency. Nonetheless, the cyt106-transformed cells appear to be fully transformed when compared with Ad2 wild type transformed cells. Unlike most other cyt mutants, cyt 106 is dominant over Ad2 wild type in mixed infections as judged by the plaque morphology in infective center assays and by the cytopathic effect. The dominant nature of the mutation may contribute to the observed transformation characteristics. Earlier we have shown that a cytocidal mutant, dl250 is partially defective in viral DNA synthesis in human KB cells. Now we show that two other cyt mutants cyt 5 (with a chain termination mutation near the C terminus) and cyt 6 (with a single amino acid substitution at position 44) are also partially defective in viral DNA synthesis in human KB cells. In contrast to these mutants, mutant cyt 106 induces normal replication of viral DNA. The DNA replication defect in mutants cyt 5 and 6 can be complemented in trans in 293 cells that constitutively express the 175R T antigen. Our results also indicate that a domain of this protein around the 44th amino acid is important for efficient viral DNA synthesis in KB cells.

Adenoviridae↗

PubMed1985

An adenovirus 2-coded tumor antigen located on the endoplasmic reticulum and nuclear envelope is required for growth of transformed cells in Ca2+-deficient media.

Rat embryo cell lines containing the adenovirus 2 E1a region together with normal or mutant forms of the N-terminal half of the E1b region (HindIII G fragment) were generated by using a dominant selection marker, neo. Biochemically transformed cells containing a nonmutated HindIII G fragment proliferated more rapidly in Ca2+-deficient media, whereas cells containing a specific deletion within the E1b-encoded, 175-amino-acid (175R) (19-kilodalton) T-antigen gene and nontransformed cells grew at a slower rate. Furthermore, transformed cells that did not express the 175R T antigen and untransformed cells could not replicate their DNA efficiently in low-Ca2+ medium. Our results suggest that Ca2+ ions may provide an important stimulus for cell proliferation in adenovirus-transformed cells through a mechanism that involves the functions of the 175R T antigen.

Adenoviruses, Human↗

PubMed1984

19-kDa tumor antigen coded by early region E1b of adenovirus 2 is required for efficient synthesis and for protection of viral DNA.

The adenovirus E1b region (mp 4.5-11.2) codes for two major tumor antigens of 53 and 19 kDa. The lp+ locus maps within the 19-kDa tumor antigen-coding region and has been shown to play an essential role in cell transformation. We have investigated the role of the 19-kDa tumor antigen during productive virus growth using a specific Ad2 mutant (dl250) lacking most of the 19-kDa tumor antigen-coding region. Mutant dl250 grows more slowly and yields about 100-fold less progeny virus than Ad2 wt in human KB cells. In cells infected with mutant dl250, viral DNA is present at only about one-half the level of that of Ad2 wt. The defect in DNA accumulation appears to be both at the level of DNA synthesis and stability of newly synthesized DNA. In mutant-infected cells, newly replicated viral as well as cellular DNA are extensively degraded during late stages of viral infection. We have mapped a class of Ad12 (highly oncogenic group A) mutants (cyt) that are nononcogenic in newborn hamsters and induce DNA degradation to the 19-kDa tumor antigen-coding region by intertypic complementation analysis. These results strongly suggest that the 19-kDa tumor antigen plays an essential role in efficient viral DNA synthesis and protection of newly replicated viral DNA against cellular nucleases in addition to its role in cell transformation and tumorigenesis.

Adenoviridae↗