The cDNA fragment of the large RNA segment of infectious bursal disease virus 002-73, when expres... more The cDNA fragment of the large RNA segment of infectious bursal disease virus 002-73, when expressed in Escherichia coli, produces precursor polyprotein (N-VP2-VP4-VP3-C), most of which is then processed to generate constituent polypeptides. Using cDNA fragments containing site-specific mutations and two monoclonal antibodies that are specific to VP2 and VP3 of mature virus particles, we demonstrated that the VP4 protein is involved in processing of the precursor polyprotein to generate VP2 and VP3 and excluded the possibility of internal initiation for the generation of VP3.
Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune... more Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune responses. Coat protein (CP) monomers of a potyvirus (Johnsongrass mosaic virus) when produced in heterologous host expression systems (Escherichia coli, yeast and insect cells) self-polymerized to produce potyvirus-like particles (PVLPs). The N- and C-terminal regions of potyvirus CP are surface-exposed and are not required for assembly. Hybrid CP monomers containing short peptides fused to their N- and/or C-termini, or large target antigens fused to the N-terminus or replacing most of the N- or C-terminal exposed regions retained the ability to assemble into hybrid PVLPs. Such chimeric PVLPs were highly immunogenic in mice and rabbits even in the absence of any adjuvant. Potyvirus CP is highly versatile in accommodating peptides or large antigens and is able to present antigens exposed on the surface of virus-like particles. This, combined with the efficiency of high level bacterial and insect cell expression systems, makes PVLPs an attractive non-pathogenic and non-replicative vaccine carrier.
Syntaxin 1A has been identified previously as a neural-cell-specific, membrane-anchored receptor ... more Syntaxin 1A has been identified previously as a neural-cell-specific, membrane-anchored receptor protein required for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Syntaxin 1A consists of 288 amino acid residues including a 265-residue N-terminal region exposed to the cytoplasm and a C-terminal hydrophobic stretch of 23 residues believed to anchor syntaxin to the plasma membrane. Using a human fat-cell library we have isolated a novel cDNA clone of syntaxin 1A containing an insert of 91 bp in codon 226. This insert and subsequent frame shift generated a cDNA that codes for a truncated protein of 260 residues without the C-terminal transmembrane domain characteristic of the syntaxin family. Analysis of the deduced amino acid sequence of the new cDNA clone, termed syntaxin 1C, showed that it was identical for the first 226 residues with the previously described neural syntaxin 1A, and diverged thereafter. The truncated protein lacked the botulinum neuro...
Insulin stimulation of glucose transport in the major insulin-responsive tissues results predomin... more Insulin stimulation of glucose transport in the major insulin-responsive tissues results predominantly from the translocation to the cell surface of a particular glucose transporter isoform, GLUT4, residing normally under basal conditions in intracellular vesicular structures. Recent studies have identified the presence of vesicle-associated membrane protein (VAMP) 2, a protein involved in vesicular trafficking in secretory cell types, in the vesicles of insulin-sensitive cells that contain GLUT4. The plasma membranes of insulin-responsive cells have also been shown to contain syntaxin 4 and the 25 kDa synaptosome-associated protein (SNAP-25), two proteins that form a complex with VAMP 2. The potential functional involvement of VAMP 2, SNAP-25 and syntaxin 4 in the trafficking of GLUT4 was assessed in the present study by determining the effect on GLUT4 translocation of microinjection of toxins that specifically cleave VAMPs or SNAP-25, or microinjection of specific peptides from VA...
Publisher Summary This chapter discusses how site-directed Transposase (Tn5) mutagenesis is used ... more Publisher Summary This chapter discusses how site-directed Transposase (Tn5) mutagenesis is used in the laboratory to identify genes essential for symbiotic nitrogen fixation in slow-growing rhizobium species. This chapter has experimented with several methods for this kind of mutagenesis and will discuss the protocol that has worked most successfully in conjunction with these organisms. The chapters also describe a method for site-directed transplacement of in vitro altered DNA sequences that should be applicable to all gram-negative bacteria. Tn5 is stably maintained in the chromosome throughout the life cycle of the rhizobium cell in the nodules of the plant. Maintenance of an extrachromosomal plasmid, such as plasmid (pVK100) under these same nonselective conditions, however, is sometimes a problem. Moreover, false-positive complementation results can occur between two Tn5 insertions that are actually within the same transcriptional unit as a result of marker exchange between the homologous Rhizobium DNA contained on the plasmid and in the chromosome. Although the advancement of the genetics of the slow-growing Rhizobium species has preceded more slowly than that of the fast-growing Rhizobium species, the techniques for mutagenesis described in this chapter should facilitate progress in this field.
In view of the enormous challenge and pressure on farmers to feed 9 billion plus people and billi... more In view of the enormous challenge and pressure on farmers to feed 9 billion plus people and billions of animals who are going to be living in our planet in 2050, new technologies must be invented, assessed and adapted. Farmer welfare and provision of resources required for their work is of paramount importance. India has benefited from Bt cotton technology and will certainly benefit from other biotech crops that have been carefully developed and assessed for consumption and environmental safety.
The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the v... more The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the viral polypeptides are present in the following order: N-VP2-VP4-VP3-C. Expression in Escherichia coli of the large segment results in the processing of the polyprotein. The expression product reacts with a virus neutralizing and protective monoclonal antibody that recognizes a conformational epitope on the surface of the virus. Different regions of the large genomic segment were deleted at defined restriction sites and the truncated fragments were ligated to various expression vectors for high-level expression in E. coli. The expressed proteins were probed with three different monoclonal antibodies that recognize epitopes encoded by different regions of the large genomic segment. These deletion mapping studies suggest that VP4 is involved in the processing of the precursor polyprotein, and the conformational epitope recognized by the virus neutralizing monoclonal antibody is present within VP2.
Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the world&... more Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the world's poultry industries, causes a severe immunodepressive disease in young chickens. Maternal antibodies are able to protect the progeny passively from IBDV infection. The gene encoding the IBDV host-protective antigen (VP2) has been cloned and expressed in yeast resulting in the production of an antigen that very closely resembles native VP2. When injected into specific pathogen free chickens a single dose of microgram quantities of the yeast derived antigen induces high titres of virus neutralizing antibodies that are capable of passively protecting young chickens from infection with IBDV.
A method that directs transplacement of in vitro altered DNA sequences to substitute the correspo... more A method that directs transplacement of in vitro altered DNA sequences to substitute the corresponding wild-type DNA sequences at the original location in the Rhizobium genome is described. The NPTII gene of transposon Tn5 which confers resistance to several antibiotics in a wide variety of organisms is used as a selectable marker on the vector. To generate DNA fragment substitution,
The cDNA fragment of the large RNA segment of infectious bursal disease virus 002-73, when expres... more The cDNA fragment of the large RNA segment of infectious bursal disease virus 002-73, when expressed in Escherichia coli, produces precursor polyprotein (N-VP2-VP4-VP3-C), most of which is then processed to generate constituent polypeptides. Using cDNA fragments containing site-specific mutations and two monoclonal antibodies that are specific to VP2 and VP3 of mature virus particles, we demonstrated that the VP4 protein is involved in processing of the precursor polyprotein to generate VP2 and VP3 and excluded the possibility of internal initiation for the generation of VP3.
Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune... more Presentation of subunit vaccines in a highly ordered aggregate form can result in enhanced immune responses. Coat protein (CP) monomers of a potyvirus (Johnsongrass mosaic virus) when produced in heterologous host expression systems (Escherichia coli, yeast and insect cells) self-polymerized to produce potyvirus-like particles (PVLPs). The N- and C-terminal regions of potyvirus CP are surface-exposed and are not required for assembly. Hybrid CP monomers containing short peptides fused to their N- and/or C-termini, or large target antigens fused to the N-terminus or replacing most of the N- or C-terminal exposed regions retained the ability to assemble into hybrid PVLPs. Such chimeric PVLPs were highly immunogenic in mice and rabbits even in the absence of any adjuvant. Potyvirus CP is highly versatile in accommodating peptides or large antigens and is able to present antigens exposed on the surface of virus-like particles. This, combined with the efficiency of high level bacterial and insect cell expression systems, makes PVLPs an attractive non-pathogenic and non-replicative vaccine carrier.
Syntaxin 1A has been identified previously as a neural-cell-specific, membrane-anchored receptor ... more Syntaxin 1A has been identified previously as a neural-cell-specific, membrane-anchored receptor protein required for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Syntaxin 1A consists of 288 amino acid residues including a 265-residue N-terminal region exposed to the cytoplasm and a C-terminal hydrophobic stretch of 23 residues believed to anchor syntaxin to the plasma membrane. Using a human fat-cell library we have isolated a novel cDNA clone of syntaxin 1A containing an insert of 91 bp in codon 226. This insert and subsequent frame shift generated a cDNA that codes for a truncated protein of 260 residues without the C-terminal transmembrane domain characteristic of the syntaxin family. Analysis of the deduced amino acid sequence of the new cDNA clone, termed syntaxin 1C, showed that it was identical for the first 226 residues with the previously described neural syntaxin 1A, and diverged thereafter. The truncated protein lacked the botulinum neuro...
Insulin stimulation of glucose transport in the major insulin-responsive tissues results predomin... more Insulin stimulation of glucose transport in the major insulin-responsive tissues results predominantly from the translocation to the cell surface of a particular glucose transporter isoform, GLUT4, residing normally under basal conditions in intracellular vesicular structures. Recent studies have identified the presence of vesicle-associated membrane protein (VAMP) 2, a protein involved in vesicular trafficking in secretory cell types, in the vesicles of insulin-sensitive cells that contain GLUT4. The plasma membranes of insulin-responsive cells have also been shown to contain syntaxin 4 and the 25 kDa synaptosome-associated protein (SNAP-25), two proteins that form a complex with VAMP 2. The potential functional involvement of VAMP 2, SNAP-25 and syntaxin 4 in the trafficking of GLUT4 was assessed in the present study by determining the effect on GLUT4 translocation of microinjection of toxins that specifically cleave VAMPs or SNAP-25, or microinjection of specific peptides from VA...
Publisher Summary This chapter discusses how site-directed Transposase (Tn5) mutagenesis is used ... more Publisher Summary This chapter discusses how site-directed Transposase (Tn5) mutagenesis is used in the laboratory to identify genes essential for symbiotic nitrogen fixation in slow-growing rhizobium species. This chapter has experimented with several methods for this kind of mutagenesis and will discuss the protocol that has worked most successfully in conjunction with these organisms. The chapters also describe a method for site-directed transplacement of in vitro altered DNA sequences that should be applicable to all gram-negative bacteria. Tn5 is stably maintained in the chromosome throughout the life cycle of the rhizobium cell in the nodules of the plant. Maintenance of an extrachromosomal plasmid, such as plasmid (pVK100) under these same nonselective conditions, however, is sometimes a problem. Moreover, false-positive complementation results can occur between two Tn5 insertions that are actually within the same transcriptional unit as a result of marker exchange between the homologous Rhizobium DNA contained on the plasmid and in the chromosome. Although the advancement of the genetics of the slow-growing Rhizobium species has preceded more slowly than that of the fast-growing Rhizobium species, the techniques for mutagenesis described in this chapter should facilitate progress in this field.
In view of the enormous challenge and pressure on farmers to feed 9 billion plus people and billi... more In view of the enormous challenge and pressure on farmers to feed 9 billion plus people and billions of animals who are going to be living in our planet in 2050, new technologies must be invented, assessed and adapted. Farmer welfare and provision of resources required for their work is of paramount importance. India has benefited from Bt cotton technology and will certainly benefit from other biotech crops that have been carefully developed and assessed for consumption and environmental safety.
The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the v... more The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the viral polypeptides are present in the following order: N-VP2-VP4-VP3-C. Expression in Escherichia coli of the large segment results in the processing of the polyprotein. The expression product reacts with a virus neutralizing and protective monoclonal antibody that recognizes a conformational epitope on the surface of the virus. Different regions of the large genomic segment were deleted at defined restriction sites and the truncated fragments were ligated to various expression vectors for high-level expression in E. coli. The expressed proteins were probed with three different monoclonal antibodies that recognize epitopes encoded by different regions of the large genomic segment. These deletion mapping studies suggest that VP4 is involved in the processing of the precursor polyprotein, and the conformational epitope recognized by the virus neutralizing monoclonal antibody is present within VP2.
Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the world&... more Infectious bursal disease virus (IBDV), a pathogen of major economic importance to the world's poultry industries, causes a severe immunodepressive disease in young chickens. Maternal antibodies are able to protect the progeny passively from IBDV infection. The gene encoding the IBDV host-protective antigen (VP2) has been cloned and expressed in yeast resulting in the production of an antigen that very closely resembles native VP2. When injected into specific pathogen free chickens a single dose of microgram quantities of the yeast derived antigen induces high titres of virus neutralizing antibodies that are capable of passively protecting young chickens from infection with IBDV.
A method that directs transplacement of in vitro altered DNA sequences to substitute the correspo... more A method that directs transplacement of in vitro altered DNA sequences to substitute the corresponding wild-type DNA sequences at the original location in the Rhizobium genome is described. The NPTII gene of transposon Tn5 which confers resistance to several antibiotics in a wide variety of organisms is used as a selectable marker on the vector. To generate DNA fragment substitution,
Uploads
Papers by MITTUR JAGADISH