PubMed Health⌕ Search

Biomedical subjects

M Padidam

Publications and source records attributed to M Padidam.

10 recordsLinked to original sources
PubMed2001

Elimination of transcriptional interference between tandem genes in plant cells.

Plant cells are commonly transformed with two or more tandemly arranged genes, but how orientation affects their expression is not well understood. We investigated the amount of transcriptional interference occurring between two adjacent genes by cloning luciferase and green fluorescent protein (GFP) genes (promoter--coding sequence--terminator) in all possible orientations and expressing the genes in tobacco protoplasts. When two genes are oriented head-to-tail (-->-->), the expression of the downstream gene was reduced 80% by the upstream gene. When two genes are oriented tail-to-tail (--><--), the expression of the upstream gene was reduced 53% by the expression of the downstream gene. There was no interference when the orientation was head-to-head (<---->). Using a chemically inducible gene expression system, we showed that the downstream gene expression was reduced 71% by the induction of an upstream gene. Inserting a mammalian transcription blocker sequence eliminated the interference between the genes in tail-to-tail orientation. The interference in the head-to-tail orientation was eliminated by inserting a 2322-bp lambda phage DNA fragment. The terminators in gene constructs did not prevent the transcriptional interference, and the interference was eliminated by designing the orientation of genes and by placing a transcription blocker or a lambda phage sequence between genes.

Bacteriophage lambda↗
PubMed1999

Possible emergence of new geminiviruses by frequent recombination.

Although exchange of genetic information by recombination plays a role in the evolution of viruses, the extent to which it generates diversity is not clear. We analyzed genomes of geminiviruses for recombination using a new statistical procedure developed to detect gene conversions. Geminiviruses (family, Geminiviridae) are a group of plant viruses characterized by a genome of circular single-stranded DNA (approximately 2700 nucleotides in length) encapsidated in twinned quasi-isometric particles. Complete nucleotide sequences of geminiviruses were aligned, and recombination events were detected by searching pairs of viruses for sequences that are significantly more similar than expected based on random distribution of polymorphic sites. The analyses revealed that recombination is very frequent and occurs between species and within and across genera. Tests identified 420 statistically significant recombinant fragments distributed across the genome. The results suggest that recombination is a significant contributor to geminivirus evolution. The high rate of recombination may be contributing to the recent emergence of new geminivirus diseases.

Evolution, Molecular↗
PubMed1999

Molecular characterization of a plant mitochondrial chaperone GrpE.

Escherichia coli DnaK (Hsp70) cooperates with DnaJ and GrpE in its essential role as a molecular chaperone. Function of mitochondrial Hsp70 (mHsp70) in protein folding and organellar import in eukaryotes is critically dependent on GrpE. We cloned two genes from tobacco (Nicotiana tabacum) BY2 cells based on peptide sequences from a purified protein. The predicted amino acid sequences of both clones resembled that of GrpE from E. coli and its homologues from eukaryotes, and a cDNA clone from Arabidopsis thaliana. One gene (Type 1) encoded a deduced protein that was identical to the purified protein while the other (Type 2) encoded a deduced protein that has 80% sequence identity to Type 1. Both tobacco and Arabidopsis thaliana GrpE homologues bound to DnaK and ATP inhibited this binding. The tobacco GrpE homologue contained a typical N-terminal mitochondrial target presequence of 64 residues and the presequence directed the green fluorescent protein to tobacco mitochondria. The tobacco GrpE homologue also associated with mHsp70 when reintroduced into BY2 protoplasts, and this association was disrupted by ATP. A three-dimensional structure for the tobacco GrpE homologue was modeled based on the X-ray structure of E. coli GrpE complexed with DnaK. The modeled structure has the same overall structure as E. coli GrpE. We propose that the tobacco GrpE homologue interacts with mHsp70 in a manner analogous to E. coli GrpE with DnaK and designate it as tobacco mitochondrial GrpE (NtmGrpE).

Amino Acid Sequence↗
PubMed1999

A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement.

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.

Bacteriophage M13↗
PubMed1999

Identification of replication specificity determinants in two strains of tomato leaf curl virus from New Delhi.

We used two strains of tomato leaf curl virus from New Delhi to investigate specificity in replication of their cognate genomes. The strains share 94% sequence identity and are referred to as severe and mild on the basis of symptoms on tomato and tobacco. Replication assays in tobacco protoplasts and plants showed that a single amino acid change, Asn10 to Asp in the N terminus of Rep protein, determines specificity for replication of the two strains based upon its interaction with the origin of replication (ori) sequences. The change of Asp10 to Asn in Rep protein of the mild strain coupled with point mutations at the 3rd and 10th nucleotides of the 13-mer binding site altered its replication ability, resulting in increased levels of virus accumulation. Similarly, changing Asn10 to Asp in Rep protein of the severe strain impaired replication of the virus and altered its severe phenotype in plants. Site-directed mutations made in ori and Asn10 of Rep protein suggested that Asn10 recognizes the third base pair of the putative binding site sequence GGTGTCGGAGTC in the severe strain.

Asparagine↗
PubMed1996

The role of AV2 ("precoat") and coat protein in viral replication and movement in tomato leaf curl geminivirus.

We analyzed various mutants of tomato leaf curl virus-India to investigate the role of ORFs AV3, AV2, and coat protein (CP) in viral replication, movement, and symptom development. The results of these studies indicate that ORF AV3 does not encode a protein. Plants inoculated with infectious DNA which contained deletions in AV2 developed very mild symptoms and accumulated only low levels of both single-stranded (ss) and double-stranded (ds) viral DNA, whereas inoculated protoplasts accumulated both ss and dsDNA to wild-type levels, showing that AV2 is required for efficient viral movement. However, both plants and protoplasts inoculated with substitution, frameshift, and other similar mutations in AV2 accumulated low levels of viral DNA. The low levels of accumulation of DNA of these mutants were apparently not due to a defect in AV2 synthesis. Mutations in the CP caused a marked decrease in ssDNA accumulation in plants and protoplasts while increasing dsDNA accumulation in protoplasts. Mutations in both AV2 and CP behaved like AV2 mutants in plants and like CP mutants in protoplasts. The results demonstrated that multiple functions provided by AV2, BV1, BC1 are essential for viral movement, and that changes in A-component virion-sense mRNA structure or translation affect viral replication.

Amino Acid Sequence↗
PubMed1995

Tomato leaf curl geminivirus from India has a bipartite genome and coat protein is not essential for infectivity.

Genomes of two isolates of tomato leaf curl geminivirus from India (ToLCV-India) have been sequenced. ToLCV-India contains A and B components, both of which are required for systemic movement and symptom development. The two isolates have 94% sequence identify but one isolate gave mild symptoms in Nicotiana benthamiana and tomato. The genome organization of ToLCV-India is similar to other whitefly-transmitted geminiviruses (WTGs) with bipartite genomes. However, it contains an additional ORF, AV3, that has not been reported for other WTGs. Its coat protein (CP) sequence is highly homologous to that of Indian cassava mosaic virus (90%). Two mutations that truncated the CP after amino acids 65 or 172 did not affect systemic movement and symptom development in either N. benthamiana or tomato. However, the symptoms caused by mutant viruses were different from those in plants infected with unmodified viruses, and plants infected with the mutants had markedly reduced amounts of single-stranded viral DNA. Comparison of sequences and other biological features of ToLCV-India with other geminiviruses showed that ToLCV-India is a distinct virus and is related to the WTGs from the Old World. It is similar to African cassava mosaic virus in its requirement for B component and dispensability of coat protein for symptom development, unlike other geminiviruses that infect tomato in the Old World. It is proposed that ToLCV-India evolved more recently as compared to other geminiviruses that infect tomato in the Old World.

Base Sequence↗
PubMed1995

Classification and identification of geminiviruses using sequence comparisons.

The genomes and ORFs of 36 geminiviruses were compared to obtain phylogenetic trees and frequency distributions of all possible pairwise comparisons with an objective to classify geminiviruses. Such comparisons show that geminiviruses form two distinct clusters of leafhopper-transmitted viruses that infect monocots (subgroup I) and whitefly-transmitted viruses that infect dicots (subgroup III), irrespective of the part of the genome considered. Of the two leafhopper-transmitted viruses that infect dicots, tobacco yellow dwarf virus has a sequence most similar to subgroup I viruses, and that of beet curly top virus differed depending upon the ORF considered. The distributions of identities within subgroups are significantly different suggesting that the taxonomic status of a particular isolate within a subgroup can be quantified. All the recognized strains of any one virus have greater than 90% sequence identity. It was observed that the 200 nucleotide intercistronic regions of geminiviruses are more variable than the remainder of the genome. The amino acid sequences of the coat protein (CP) of subgroup III viruses are more conserved than the remainder of the genome. However, a short N-terminal region (60-70 amino acids) of the CP is more variable than the rest of the CP sequence and is a close representation of the genome. PCR primers based on conserved sequences can be used to clone and sequence the N-terminal sequences of the CP of the geminiviruses; this sequence is sufficient to classify a virus isolate. A possible taxonomic structure for geminiviruses is proposed after considering the sequence comparisons and biological properties.

Amino Acid Sequence↗