Biomedical subjects

T Subramanian

Publications and source records attributed to T Subramanian.

13 recordsLinked to original sources

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↗

PubMed1977

Comparison of antibody-dependent cellular cytotoxicity and complement-dependent antibody lysis of herpes simplex virus-infected cells as methods of detecting antiviral antibodies in human sera.

An antibody-dependent cellular cytotoxicity (ADCC) assay was used to detect antibodies to the herpes simplex viruses in humans sera. The assay utilized the release of 51Cr from BHK-21 cells infected with the viruses, hamster peritoneal exudate cells as effector cells, and antiviral antibodies in human sera. The technique was found to be far more sensitive than complement-dependent antibody lysis of infected cells and virus neutralization. The ADCC assay was useful in detecting antibodies in sera that had low titers or no antibodies detectable by other methods. In a sample of 100 sera from university students, 40 were positive by complement-dependent lysis whereas 73 were positive by ADCC. Of 400 sera from women with cervical cancer, 17 did not have detectable antibodies by microneutralization or complement-dependent lysis; however, all sera were positive by ADCC, suggesting that all of the women had been infected in the past with one or both types of herpes simplex virus.

Antibodies, Viral↗