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T Chakraborty

Publications and source records attributed to T Chakraborty.

At least 127 records · Page 7Linked to original sources
PubMed1992

Expression of bacterial cytotoxin genes in mammalian target cells.

We have studied the expression of the gene fragments encoding the enzymatically active portion of three bacterial cytotoxins: exotoxin A (ETA) of Pseudomonas aeruginosa, and pertussis toxin (PT) and adenylate cyclase toxin (CYA) of Bordetella pertussis, in sensitive mammalian target cells. Expression of active ETA and CYA was lethal to the producing cells and stable transfectants of Cos-1 cells containing the corresponding genes could not be obtained. The expression of the PTS1 subunit was tolerated by the producing mammalian cells. Since PT is cytotoxic because of ADP-ribosylation of G-proteins, we assume that the endogenously expressed PTS1 may not find the cellular target G proteins or PTS1 alone may not be sufficient for ADP-ribosylation of these proteins in vivo.

ADP Ribose Transferases↗
PubMed1992

Suitability of the prfA gene, which encodes a regulator of virulence genes in Listeria monocytogenes, in the identification of pathogenic Listeria spp.

The pathogenesis of listerial infections is complex and involves a number of virulence factors expressed by virulent Listeria species. We have recently described a regulator gene, prfA, that positively regulates the expression of a number of virulence factors in Listeria monocytogenes. When the prfA gene was used as a DNA probe, we found it to be extremely specific for the pathogenic species L. monocytogenes. No reaction was obtained with strains of all other species of this genus. By using this information, an oligonucleotide primer pair was developed that specifically amplifies the prfA gene in L. monocytogenes strains of all known serotypes.

Base Sequence↗
PubMed1992

Coordinate regulation of virulence genes in Listeria monocytogenes requires the product of the prfA gene.

The prfA gene of Listeria monocytogenes encodes a protein that activates transcription of the listeriolysin gene (lisA). In order to explore the role of the prfA gene product in the pathogenesis of listerial infection, we constructed a site-directed insertion mutation in prfA by the chromosomal integration of a novel suicide vector containing a portion of the prfA coding region. This mutation not only transcriptionally silenced the listeriolysin (lisA) gene but also abrogated production of specific RNA transcripts corresponding to the phosphatidylinositol-specific phospholipase C (pic) and metalloprotease (mpl) genes, two further virulence gene products expressed only by pathogenic Listeria strains. The strain was also found to be avirulent when tested in a mouse model of listerial infection. The concomitant loss of multiple characteristics such as production of LisA, Pic, Mpl, and loss of virulence in a mouse infection model is the result of a mutation in a single gene and demonstrates that the prfA gene product is a positive regulator of multiple virulence determinants in L. monocytogenes.

Animals↗
PubMed1992

The expression of virulence genes in Listeria monocytogenes is thermoregulated.

The expression of listeriolysin, a major virulence factor of the gram-positive facultative intracellular pathogen Listeria monocytogenes, is positively regulated by a transcriptional activator, the prfA gene product. We had previously shown that mutations within the prfA gene lead to loss of listeriolysin production. In this communication, the regulation of expression of listeriolysin by a specific environmental condition, namely, temperature, was studied in wild-type strains of Listeria monocytogenes. We found that expression of the hemolysis phenotype was thermoregulated. A lisA::lacZ fusion was constructed, and its expression in the wild-type strain was studied at various growth temperatures. The results showed that the fusion beta-galactosidase activity was expressed only when cultures were grown at temperatures above 30 degrees C. This activity could be either specifically repressed or induced, depending on growth temperature. No change in activity was detected in a strain harboring a control beta-galactosidase fusion at the various growth temperatures tested. Northern (RNA) blot analysis of lisA-specific RNA transcripts showed that thermoregulation is manifested at the level of transcription. We also found that the transcription of other PrfA-regulated virulence genes in L. monocytogenes was similarly affected by growth temperature. Hence, as in other facultative intracellular pathogens, Shigella and Yersinia spp., temperature is an important cue in the induction of expression of virulence genes in L. monocytogenes. Our studies revealed that a higher level of regulation is imposed on the PrfA-mediated activation of virulence genes in pathogenic L. monocytogenes.

Bacterial Toxins↗
PubMed1992

The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription.

Myogenin is a skeletal muscle-specific transcription factor that can activate myogenesis when introduced into a variety of nonmuscle cell types. Activation of the myogenic program by myogenin is dependent on its binding to a DNA sequence known as an E box, which is associated with numerous muscle-specific genes. Myogenin shares homology with MyoD and other myogenic regulatory factors within a basic region and a helix-loop-helix (HLH) motif that mediate DNA binding and dimerization, respectively. Here we show that the basic region-HLH motif of myogenin alone lacks transcriptional activity and is dependent on domains in the amino and carboxyl termini to activate transcription. Analysis of these N- and C-terminal domains through creation of chimeras with the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that they act as strong transcriptional activators. These transcription activation domains are dependent for activity on a specific amino acid sequence within the basic region, referred to as the myogenic recognition motif (MRM), when an E box is the target for DNA binding. However, the activation domains function independent of the MRM when DNA binding is mediated through a heterologous DNA-binding domain. The activation domain of the acidic coactivator VP16 can substitute for the myogenin activation domains and restore strong myogenic activity to the basic region-HLH motif. Within a myogenin-VP16 chimera, however, the VP16 activation domain also relies on the MRM for activation of the myogenic program. These findings reveal that DNA binding and transcriptional activation are separable functions, encoded by different domains of myogenin, but that the activity of the transcriptional activation domains is influenced by the DNA-binding domain. Activation of muscle-specific transcription requires collaboration between the DNA-binding and activation domains of myogenin and is dependent on events in addition to DNA binding.

Amino Acid Sequence↗
PubMed1992

Analysis of the myogenin promoter reveals an indirect pathway for positive autoregulation mediated by the muscle-specific enhancer factor MEF-2.

Transcriptional cascades that specify cell fate have been well described in invertebrates. In mammalian development, however, gene hierarchies involved in determination of cell lineage are not understood. With the recent cloning of the MyoD family of myogenic regulatory factors, a model system has become available with which to study the dynamics of muscle determination in mammalian development. Myogenin, along with other members of the MyoD gene family, possesses the apparent ability to redirect nonmuscle cells into the myogenic lineage. This ability appears to be due to the direct activation of an array of subordinate or downstream genes which are responsible for formation and function of the muscle contractile apparatus. Myogenin-directed transcription has been shown to occur through interaction with a DNA consensus sequence known as an E box (CANNTG) present in the control regions of numerous downstream genes. In addition to activating the transcription of subordinate genes, members of the MyoD family positively regulate their own expression and cross-activate one another's expression. These autoregulatory interactions have been suggested as a mechanism for induction and maintenance of the myogenic phenotype, but the molecular details of the autoregulatory circuits are undefined. Here we show that the myogenin promoter contains a binding site for the myocyte-specific enhancer-binding factor, MEF-2, which can function as an intermediary of myogenin autoactivation. Since MEF-2 can be induced by myogenin, these results suggest that myogenin and MEF-2 participate in a transcriptional cascade in which MEF-2, once induced by myogenin, acts to amplify and maintain the myogenic phenotype by acting as a positive regulator of myogenin expression.

Amino Acid Sequence↗
PubMed1991

A myogenic factor from sea urchin embryos capable of programming muscle differentiation in mammalian cells.

Using the basic helix-loop-helix domain of the myogenic factor myogenin as a probe, we identified a clone from a sea urchin cDNA library with considerable sequence similarity to the vertebrate myogenic factors. This cDNA, sea urchin myogenic factor 1 (SUM-1), transactivated a muscle creatine kinase-chloramphenicol acetyltransferase reporter gene in 10T1/2 fibroblasts to a level comparable to that of the vertebrate myogenic factors. In addition, bacterially expressed beta-galactosidase-SUM-1 fusion protein interacted directly with the kappa E-2 site in the muscle creatine kinase enhancer core as assayed by electrophoretic mobility shift assays. Stably transfected SUM-1 activated the muscle differentiation program and converted 10T1/2 cells from fibroblasts to myotubes. In sea urchin embryos, SUM-1 RNA was not detected before gastrulation. It accumulated to its highest levels during the prism stage when myoblasts were first detected by myosin immunostaining and then diminished as myocytes differentiated. SUM-1 protein was localized in secondary mesenchyme cells when they could first be identified as muscle cells by myosin immunostaining. These results implicate SUM-1 as a regulatory factor involved in the early decision of a pluripotent secondary mesenchyme cell to convert to a myogenic fate. SUM-1 is an example of an invertebrate myogenic factor that is capable of functioning in mammalian cells.

Amino Acid Sequence↗
PubMed1991

Mutagenesis of the myogenin basic region identifies an ancient protein motif critical for activation of myogenesis.

Myogenin is a muscle-specific nuclear factor that acts as a genetic switch to activate myogenesis. Myogenin, MyoD, and a growing number of proteins implicated in transcriptional control share sequence homology within a basic region and an adjacent helix-loop-helix motif. Here we identify by site-directed mutagenesis a 12-amino acid subdomain of the myogenin basic region essential for binding of DNA and activation of myogenesis. The basic region of the widely expressed helix-loop-helix protein E12 is conserved at 8 of these 12 residues and can mediate DNA binding when placed in myogenin, but it cannot activate myogenesis. Replacement of each of the four nonconserved residues of the myogenin basic region with the corresponding residues of E12 reveals two adjacent amino acids (Ala86-Thr) that can impart muscle specificity to the basic region. These residues are specific to, and conserved in, the basic regions of all known myogenic helix-loop-helix proteins from Drosophila to man, suggesting that they constitute part of an ancient protein motif required for activation of the myogenic program.

Amino Acid Sequence↗
PubMed1991

Studies on aerolysin and a serine protease from Aeromonas trota sp. nov.

Hybridization of 257 mesophilic aeromonads revealed that the aerolysin gene is present in virtually all strains irrespective of origin of isolation. A probe comprising the promotor region was specific for a species now defined as Aeromonas trota sp. nov. Finally, isolation of a serine protease that is concomitantly expressed with aerolysin is described.

Aeromonas↗
PubMed1991

Differential trans-activation of a muscle-specific enhancer by myogenic helix-loop-helix proteins is separable from DNA binding.

The muscle creatine kinase (MCK) enhancer was used as a target to study the specificity of DNA binding and trans-activation by members of the helix-loop-helix (HLH) family of myogenic regulatory factors, MyoD1, myogenin, myf-5, and MRF4. Whereas all four myogenic factors bound with similar affinities to the MCK enhancer in the presence of the widely expressed HLH protein E12, only MyoD1, myogenin, and myf-5 efficiently trans-activated the enhancer in transiently transfected 10T1/2 and 3T3 cells. That MRF4 binds the MCK enhancer without activating transcription suggests that domains in addition to those required for DNA binding are important for transcriptional activation and supports the notion that the different members of the HLH family of myogenic regulatory factors may selectively regulate unique sets of muscle-specific genes.

Cells, Cultured↗
PubMed1991

Gene disruption by plasmid integration in Listeria monocytogenes: insertional inactivation of the listeriolysin determinant lisA.

A plasmid integration technique was developed for insertional inactivation of chromosomal Listeria monocytogenes genes. A Listeria-Escherichia coli shuttle vector (pLSV1) was constructed which carried the temperature-sensitive gram-positive replication origin from plasmid pTV32(Ts). An internal fragment of the listeriolysin gene (lisA) was cloned into pLSV1 to create pLSV2. In L. monocytogenes pLSV2 transformants, plasmid pLSV2 integrated into the L. monocytogenes chromosome at a frequency of 2 x 10(-3) via lisA homology and these cells could be selected at 42 degrees C using a plasmid-encoded erythromycin resistance. Plasmid integration resulted in disruption of the lisA gene, production of a truncated, immunologically cross-reactive listeriolysin protein and loss of the hemolytic phenotype. An improved Listeria protoplast transformation method is also described which facilitates genetic manipulation of Listeria species.

Bacterial Toxins↗
PubMed1991

Studies on the pathogenicity of Listeria monocytogenes.

The characterization of mutants of Listeria monocytogenes with reduced virulence properties is described. Reduction in the amount of the extracellular protein p60 (encoded by the ipa gene) leads to cell filaments with impaired invasiveness. Mutants which cannot synthesize listeriolysin are still invasive but unable to survive within phagocytic cells. One type of listeriolysin-negative mutants is defective in the synthesis of a positive regulatory element PrfA which coordinately regulates the listeriolysin gene (lisA) together with several other genes, including those for a phosphatidylinositol-specific phospholipase and a metalloprotease.

Antigens, Bacterial↗
PubMed1991

Listeriolysin negative mutants of Listeria monocytogenes specifically stimulate T-lymphocytes mediating protection and granulomatous inflammation.

The ability of several listeriolysin O negative mutants of the virulent EGD strain of Listeria monocytogenes to activate specific T cell responses in vitro and in vivo was determined. A T cell line and a derived clone specific for Listeria monocytogenes, strain EGD, which are able to adoptively transfer protection and granuloma formation were examined. Specificity testing showed no difference between listeriolysin positive and negative strains to induce proliferation of the T cell lines and clones. Similar results were obtained when we examined T cell mediated granuloma formation in the livers of mice previously immunized with viable bacteria. Granulomatous inflammation could be elicited by i.v. application of heat killed bacteria of listeriolysin positive as well as of negative bacteria. Thus, the expression of the heat induced 60KD listeriolysin protein is not a prerequisite for the stimulation of Listeria-specific T lymphocytes mediating protection and granuloma formation.

Animals↗
PubMed1991

Proteins of the nuclear factor-1 family act as an activator of the late promoter in human polyomavirus BK in vitro.

The cis-acting elements for the early and late promoters, as well as the enhancer in the prototype strains of human polyomavirus BK (BKV) are located within a 500 bp intergenic region. We previously studied the specificity of protein binding in this region in vitro and showed that the interaction of proteins of the nuclear factor-1 (NF-1) family is crucial for early promoter activity. We have now extended our study to the BKV late promoter. We show that the late promoter activity in HeLa cell extracts is poor compared to the activity of the early promoter. Using a high template to protein ratio, multiple start sites were detected by primer extension analysis. DNase I protection experiments revealed the presence of three NF-1 binding sites in the late side, in addition to those identified previously in the 68 bp repeats and C element. Competition transcription assays using binding sites for NF-1, AP-1, Sp-1 and a complete 68 bp repeat indicated that only the 68 bp repeat and the NF-1 binding site competed significantly with the late promoter activity. A point mutation in the NF-1 binding site, which destroys the ability of the oligonucleotide to bind NF-1, also impaired its capacity to compete with the late promoter. The ability of NF-1 to activate both the early and late promoters suggests that the proteins of this family act as a bidirectional transcriptional activator in this virus.

BK Virus↗
PubMed1991

Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese.

The polymerase chain reaction (PCR) amplification technique was investigated as a tool for direct detection of Listeria monocytogenes in soft cheeses. Different sets of oligonucleotide primers were used, and parts of the L. monocytogenes Dth 18-gene could be amplified specifically when either a plasmid vector carrying the cloned gene or chromosomal DNA was used a template. The detection limit for L. monocytogenes in dilutions of pure cultures was between 1 and 10 colony-forming units. In extracts from soft cheeses containing L. monocytogenes DNA, the amplification was strongly inhibited. This inhibition could be reduced by an additional purification step. Despite this the detection limit showed a large variation, depending on the brand of cheese used. In some cheeses 10(3) cfu/0.5g could be visualized whereas in others the presence of 10(8) cfu/0.5 g did not yield a detectable quantity of amplified product.

Base Sequence↗