Search PubMed⌕ Search

Biomedical subjects

G Chinnadurai

Publications and source records attributed to G Chinnadurai.

At least 91 records · Page 5Linked to original sources
PubMed1987

Activation of the adenovirus 2 protein IX promoter by DNA replication in a transient expression assay.

We have studied the effect of template replication on transcriptional activation of the promoters for the adenoviral protein IX and E1a genes using a short-term transient expression assay. DNA replication mediated modulation of the transcriptional activity of these promoters was monitored using plasmids with limited replication capability conferred by the SV40 minimal origin of DNA replication in the monkey kidney cell line, COS M6. Our results indicate a substantial increase in protein IX promoter directed transcription in a dosage-independent manner as a consequence of the DNA replication process. In contrast, the transcriptional activity of the adenovirus 2 E1a promoter was not significantly altered. In addition to this DNA replication-mediated effect, transcription from the protein IX promoter was positively regulated by the adenovirus E1a 13S and E4 gene products but was repressed by the E1a 12S gene product to a limited extent. The evidence suggests that the effect of template replication on the transcriptional activity of plasmid-borne protein IX and E1a gene promoters in this transient expression system closely mimics the situation on the viral chromosome.

Adenovirus Early Proteins↗
PubMed1987

Relationship between the transforming and transcriptional regulatory functions of adenovirus 2 E1a oncogene.

The E1a region of adenoviruses encodes two early proteins of 289 amino acids (289R) and 243R from two differentially spliced mRNAs of 13 and 12 S, respectively. These E1a proteins of adenoviruses are multifunctional and have been shown to play an essential role in cellular immortalization and transformation. The E1a gene is also known to regulate the expression of certain viral and cellular genes in a positive or negative manner. To identify the domains of the E1a proteins required for their transformation and transcriptional regulatory functions, we have constructed and analyzed several E1a mutations. A region located between amino acid residues 125 and 127 appears to be essential for cell transformation in cooperation with both E1b and the activated cellular oncogene, T24 Har-ras. Mutation at this region does not affect the ability of E1a to trans-activate the Ad2 early E2 promoter significantly. Our experiments have not revealed the domain of E1a most essential for trans-repression. However, the region between amino acid residues 125 and 127 appears to have a small effect on E1a-mediated repression of the immunoglobulin heavy-chain and polyoma enhancers but has no significant effect on the SV40 enhancer. From our results, it appears that the transformation function of E1a can be dissociated from its transcriptional regulatory functions.

Adenovirus Early Proteins↗
PubMed1987

Functional relatedness between the E1a and E1b regions of group C and group D human adenoviruses.

The functional relatedness of the transforming genes (E1a and E1b) of adenovirus type 9 (group D) which induces mammary tumors in rats and those of the non-tumorigenic adenoviruses, Ad2 and Ad5 (group C) was examined. Transfection of established rat embryo cells with a DNA segment containing the E1a and E1b regions of Ad9 resulted in efficient transformation; similar results have been shown for group A, B and C Ads. In contrast to Ads of group A, B and C, Ad9 DNA containing the E1 region or the entire viral genome was unable to transform primary baby rat kidney (BRK) cells. The functional relatedness of genes encoded within the E1 region was compared using a mutant complementation assay in which various group C mutants defective in the entire E1 region or in the E1a or E1b regions alone as well as mutants defective exclusively within the 19K or 58K T antigens coding regions of E1b were coinfected with wild type (wt) Ad9 and tested for group C mutant DNA replication, virus production, or expression of early and late genes. These studies have shown that a defect in the entire E1 region of Ad2 could only be complemented poorly by Ad9; our earlier studies have shown that coinfection with Ad12 (group A) or Ad7 (group B) resulted in efficient complementation (Brusca and Chinnadurai (1981) J. Virol. 39, 300-305). Further analysis indicated that a defect in the E1a region could be complemented by the group D E1a region. The level of E1a complementation as judged by mutant DNA replication and activation of expression of mutant early viral genes was about one-fourth to one-fifth the level in 293 cells that constitutively express Ad5 E1a and E1b regions. Our results indicate that a defect in the E1b 19K T antigen, which leads to degradation of intracellular DNA in infected cells, could be complemented by the group D protein. However, a defect in the E1b 58K T antigen could not be efficiently complemented by the group D protein. Coinfection of group C mutants defective in the 58K T antigen and Ad9 wt did not lead to an increase in the mutant viral production. Furthermore, in cells coinfected with the 58K T antigen mutants and Ad9 wt there was a large reduction in the accumulation of group C late cytoplasmic RNA. The observed complementation defect of Ad9 in supporting multiplication of group C mutants defective in the entire E1 region may therefore be a cumulative effect of both E1a and E1b regions.

Adenovirus Early Proteins↗
PubMed1987

Adenovirus transforming 19-kD T antigen has an enhancer-dependent trans-activation function and relieves enhancer repression mediated by viral and cellular genes.

The adenovirus E1b region codes for two major tumor antigens of 19 kD and 55 kD, which are important for cell transformation. Our results indicate that the 19-kD T antigen possesses two enhancer-regulatory functions. It can trans-activate enhancer-linked promoters and relieve enhancer repression mediated by viral and cellular repressors. The 19-kD activation function enhances expression from different promoters linked to SV40, Py, Ela, and immunoglobulin heavy-chain enhancers. Enhancer activation by the 19-kD protein appears to be cell type-specific, since the heavy-chain and SV40 enhancers were not trans-activated in myeloma cells whereas the same enhancers were trans-activated in fibroblasts. The 19-kD enhancer activation function appears to be dominant over the enhancer repression function of E1a, since in cells expressing the 19-kD protein there is no significant repression despite a large increase in E1a expression. The 19-kD T antigen activates the Py enhancer in undifferentiated F9 cells indicating that the activation function of E1b masks enhancer repression by an "E1a-like" cellular gene product. The enhancer activation function of the 19-kD T antigen may be important for cell transformation and cell differentiation.

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

Evidence that a second tumor antigen coded by adenovirus early gene region E1a is required for efficient cell transformation.

The expression of the adenovirus (Ad) early coding region 1a (E1a) is required for virus-induced cell transformation and for the activation of other viral early genes and some cellular genes. Two overlapping early mRNAs of 13S and 12S that are transcribed from this region code for a 289-amino acid protein and a 243-amino acid protein, respectively. Earlier studies have shown that the 289-amino acid protein is essential for cell transformation. We have constructed an Ad type 2 (Ad2) deletion mutant (dl231) in which the intervening sequence for the 13S mRNA is precisely removed. Mutant dl231 is completely viable in human KB cells and produces normal amounts of 13S mRNA but much reduced amounts of a defective 12S mRNA. Mutant dl231 induces focal transformation of established rat embryo fibroblasts at a frequency one-fifth to one-half that of wild-type virus. However, the transformed cells are defective in their ability to form anchorage-independent colonies on semisolid medium. Therefore, our results demonstrate that the 243-amino acid protein is required for full transformation of rat embryo cells.

Adenoviruses, Human↗
PubMed1985

Immortalization of rat embryo fibroblasts by an adenovirus 2 mutant expressing a single functional E1a protein.

Expression of the adenovirus E1a and E1b genes is required for transformation of nonpermissive rodent cells. Differential splicing of the E1a precursor RNA molecules results in the production of two early mRNAs, 13S and 12S, which encode a 289-amino-acid-residue (289R) and 243R protein, respectively. Previously we constructed a mutant virus, dl231, which can only produce normal 289R protein from the E1a gene. In this report we demonstrate that dl231 induced focal transformation of primary rat embryo fibroblasts at 20% of the level of wild-type virus. dl231 transformants were immortalized and produced normal levels of E1a 13S and E1b mRNAs but only minute levels of defective E1a 12S mRNA. These transformants only minimally expressed the transformation phenotype and were similar to untransformed cells. Unlike wild-type transformants, they had a more fibroblastic morphology, were contact inhibited, grew to only low saturation density, and were limited in their ability to grow in an anchorage-independent manner in soft agar. We conclude that the 289R E1a protein can mediate immortalization of primary cells and that the 243R E1a protein is required to elicit the full transformation phenotype.

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↗
PubMed1984

Efficient transformation of rat 3Y1 cells by human adenovirus type 9.

Human adenovirus type 9 (group D) induces mammary fibroadenomas in female rats at a high frequency. Ad9 has been used to transform an established rat embryo fibroblast cell line, 3Y1. These cells were transformed very efficiently by Ad9 and the frequency of transformation was about 16 times higher than that of adenovirus type 2 (group C). Three transformed cell lines (92.2, 93.1, and 96.2) were analyzed for the content and expression of viral DNA sequences. The Ad9-transformed cell lines contained about 7 to 30 copies of integrated viral DNA. It was found that all or almost all of the viral genome was integrated in a linear form and probably exists as tandem repeats in a head-to-tail orientation. In this respect, Ad9-transformed cells resemble Ad12-transformed cells which also contain integrated nearly complete copies of the viral genome. The Ad9-transformed cells have been shown to express the E1A and E1B regions at the RNA level.

Adenoviruses, Human↗
PubMed1984

Adenovirus cyt+ locus, which controls cell transformation and tumorigenicity, is an allele of lp+ locus, which codes for a 19-kilodalton tumor antigen.

The early region E1b of adenovirus type 2 (Ad2) codes for two major tumor antigens of 53 and 19 kilodaltons (kd). The adenovirus lp+ locus maps within the 19-kd tumor antigen-coding region (G. Chinnadurai, Cell 33:759-766, 1983). We have now constructed a large-plaque deletion mutant (dl250) of Ad2 that has a specific lesion in the 19-kd tumor antigen-coding region. In contrast to most other Ad2 lp mutants (G. Chinnadurai, Cell 33:759-766, 1983), mutant dl250 is cytocidal (cyt) on infected KB cells, causing extensive cellular destruction. Cells infected with Ad2 wt or most of these other Ad2 lp mutants are rounded and aggregated without cell lysis (cyt+). The cyt phenotype of dl250 resembles the cyt mutants of highly oncogenic Ad12, isolated by Takemori et al. (Virology 36:575-586, 1968). By intertypic complementation analysis, we showed that the Ad12 cyt mutants indeed map within the 19-kd tumor antigen-coding region. The transforming potential of dl250 was assayed on an established rat embryo fibroblast cell line, CREF, and on primary rat embryo fibroblasts and baby rat kidney cells. On all these cells, dl250 induced transformation at greatly reduced frequency compared with wt. The cells transformed by this mutant are defective in anchorage-independent growth on soft agar. Our results suggest that the 19-kd tumor antigen (in conjunction with E1a tumor antigens) may play an important role in the maintenance of cell transformation. Since we have mapped the low-oncogenic or nononcogenic Ad12 cyt mutants within the 19-kd tumor antigen-coding region, our results further indicate that the 19-kd tumor antigen also directly or indirectly plays an important role in tumorigenesis of Ad12. Our results show that the cyt+ locus is an allele of the lp+ locus and that the cyt phenotype may be the result of mutations in specific domains of the 19-kd tumor antigen.

Adenoviruses, Human↗
PubMed1983

Hinf family: a novel repeated DNA family of the human genome.

The isolation of a mutant adenovirus carrying an insertion of cellular DNA has led to the identification of a new family of human repetitive sequences, which are found tandemly arranged in the genome. The sequence of the viral insert resembles that of eukaryotic transposable elements.

Adenoviruses, Human↗
PubMed1983

Structure and reversion of an adenovirus type 2 mutant containing a duplication of the left end of the genome at the right end.

A mutant (dp 201) of human adenovirus type 2 has been isolated that has the left 3% (1110 bp) of the genome duplicated at the right terminus. The structure of the mutant genome was studied by restriction endonuclease analysis and by direct DNA sequence analysis. The duplication has resulted in a deletion of 10 bp of the right inverted terminal repeat (ITR) at the junction of right and left end sequences. This internal ITR is not active in initiation or termination of DNA synthesis. Two subclones of dp 201 that bear a deletion between map positions 2.1 and 6.8 encompassing early gene blocks E1a and E1b were also isolated. One of these isolates (dl 201.1) retains the left end duplication and the other isolate (dl 201.2) lacks the left end duplication and is an apparent revertant of dl 201.1. The original mutant dp 201 also reverts to a wild type sequence at a low rate. Since 10 base pairs of the right ITR are deleted in the mutant, reversion must involve a repair mechanism using one of the two complete left end ITRs in the mutant genome. The mechanisms by which this reversion may occur are discussed.

Adenoviruses, Human↗
PubMed1983

Adenovirus 2 Ip+ locus codes for a 19 kd tumor antigen that plays an essential role in cell transformation.

Adenovirus 2 large plaque (Ip) mutants produce large clear plaques on human KB cells. These mutants are shown to be defective in inducing transformation of the established rat embryo cell line 3Y1. The Ip mutation was localized within one of the two transforming early gene blocks, E1b (map position 4.5 to 11.2) which codes for two major T antigens of 53 kd and 19 kd by marker transfer. The mutational defects in mutants Ip3 and Ip5 were analyzed by DNA sequence analysis and by analysis of viral E1 proteins. These results reveal that Ip3 and Ip5 mutations map within the 19 kd tumor antigen coding region. Mutant Ip3 has a single base pair change at the N terminus of 19 kd polypeptide, resulting in the substitution of valine for alanine. Mutant Ip5 has two mutational changes, one of which results in the substitution of tyrosine for aspartic acid near the N-terminal region. The second mutation changes the termination codon into a leucine codon, increasing the size of the 19 kd tumor antigen. These results provide direct genetic evidence for an essential role of the 19 kd tumor antigen in cell transformation and indicate that the N-terminal region of the 19 kd tumor antigen is an essential function domain for the induction of cell transformation.

Adenoviruses, Human↗
PubMed1981

Method for introducing site-specific mutations into adenovirus 2 genome: construction of a small deletion mutant in VA-RNAI gene.

We have developed a method for introducing mutations into the adenovirus type 2 genome at predetermined sites. Specific mutations are introduced into segments of the viral genome cloned in bacteria by using plasmid vectors. The chimeric DNA is used to construct viral mutants by cotransfection with two viral DNA segments derived from both ends of the viral genome, each of which has overlapping sequence homology with the cloned viral DNA segment. To illustrate this procedure, we cloned a large restriction fragment [EcoRI fragment A, map position (mp) 0--58.5] by using plasmid vector pBR322, and a small deletion mutation was introduced at the BamHI cleavage site located within the VA-RNAI gene (mp 29 at residue +75 on VA-RNAI gene). The mutated DNA was then used to construct viral mutants by cotransfection into human cells with the adenovirus type 2 DNA--protein complex digested with Sal I. In vivo recombination occurred via overlapping sequences between the cloned EcoRI fragment A and Sal I-digested DNA--protein complex at two sites, mp 0--25 (i.e., within Sal I fragment B) and mp 45--58.5 (i.e., overlapping sequences between Sal I fragment A and EcoRI fragment A), generating infectious DNA molecules with intact ends. The viral mutant grows as well as the wild type in KB cells and induces the synthesis of smaller VA-RNAI but normal-size VA-RNAII.

Adenoviruses, Human↗
PubMed1981

Transforming genes among three different oncogenic subgroups of human adenoviruses have similar replicative functions.

We have examined the functional similarity of the transforming genes for replicative functions among three different subgroups of human adenoviruses (A, B, and C), using mutant complementation as an assay. A host range deletion mutant (dl201.2) of Ad2 (nononcogenic subgroup C) lacking about 5% of the viral DNA covering two early gene blocks (E1a and E1b) involved in cellular transformation was isolated and tested for its ability to replicate in nonpermissive KB cells in the presence of Ad7 (weakly oncogenic group B) or ad12 (highly oncogenic group A). The complementation of the mutant defect was demonstrated by cleaving the viral DNA extracted from mixed infected cells or the DNA extracted from purified virions from mixed infected cells with restriction endonuclease BamHI, which produces a different cleavage pattern with the DNA of each serotype. It was found that the defects in E1a plus E1b of dl201.2 could be complemented by Ad7 and Ad12, indicating that these genes in Ad2, Ad7, and Ad12 have similar functions during productive infection.

Adenoviruses, Human↗