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A N Shiian

Publications and source records attributed to A N Shiian.

6 recordsLinked to original sources

[A single reverse mutation in the 126/183-kda replicase gene of the attenuated tomato strain V-69 of tobacco mosaic virus increases the virus pathogenicity].

To determine the roles of the mutations that were earlier found in the structure of the attenuated tomato strain V-69 of tobacco mosaic virus (TMV), the nucleotide sequences of genomic RNAs of its four pathogenic revertants have been identified and analyzed. The comparison of the structures of viral genomes has demonstrated that all revertants studied have a single reverse mutation at position 1654 relative to the parental TMV strain V-69. This reversion has been introduced into the plasmid construction harboring a full-length cDNA copy of the TMV strain V-69 RNA, and the infective transcripts synthesized in vitro have been tested on TMV-sensitive tomato and tobacco plants. The tests have confirmed that the single reversion at position 1654, which changes the amino acid residue at position 528 of the 126-kDa and 183-kDa proteins of the viral replicase is responsible for the increase in the pathogenicity of revertants compared to the TMV V-69 strain. The mutation at position 1654 of the viral genome may be supposed to be the main factor of the attenuation of TMV strain V-69.

Base Sequence↗

[Construction of a full-length cDNA of tobacco mosaic virus strain V-69 genome].

The genome of the tobacco mosaic virus (TMV) strain V-69 was sequenced by us earlier. The comparison of its nucleotide sequence with those of well-known attenuated strains (using published data) demonstrated considerable homology between strain V-69 and tomato strain L of TMV. In this study, full-length cDNA copy of TMV strain V-69 genomic RNA has been cloned in vector pBS(+) under the control of the T7 promoter and with an SmaI site at the 3'-terminus. The in vitro transcription of this construction with the use of phage T7 RNA polymerase in the presence of a cap analog (m7GpppG) has yielded an infectious RNA. This RNA has induced the same symptoms as those caused by TMV strain V-69 in infected indicator plants. Three point mutations inactivating one of two HindIII sites have been introduced into the cDNA. The absence of the HindIII site at position 1449 of the viral nucleotide sequence and the corresponding mutations serve as markers of the construction obtained. The full-length cDNA copy of the TMV strain V-69 RNA can be used both as a research tool and for constructing a vector for transit gene expression.

Cloning, Molecular↗

[The primary structure of a vaccine strain of tobacco mosaic virus V-69].

A random set of cDNA fragments were synthesized on genomic RNA of TMV vaccine strain V-69, using random primers and reverse transcriptase. Following synthesis of double-stranded cDNA, they were cloned into the pUC-19 plasmid; and 28 clones were sequenced (insert size 100-500 bp). High nucleotide sequence homology of V-69 (more than 95%) was shown only with tomato strain TMV-L [1]. Sequenced clones represent 54% of the genome (50% of the replicase gene, 98% of the transport protein gene, and 60% of the coat protein gene). In this genome region, 24 base substitutions were revealed, as compared to the wild-type TMV-L sequence. Six base substitutions resulted in changes in corresponding amino acid codons. No substitutions coincided with those discovered in the related TMV vaccine strain L11A [2], while two substitutions in the replicase gene were identical to those found in TMV strain Lta1 [3], which is capable of overcoming protection in tomatoes with the resistance gene Tm-1.

Amino Acid Sequence↗

[Isolation and characteristics of compound transposons Tn1000::Tn5].

Two types of compound transposons were derived. In the first case, transposon Tn5 is inserted into the gene responsible for Tn1000 transposase synthesis. In the other, Tn5 is inserted into the region near the left end of Tn1000, where no functionally significant genes were found. It is known that translocation of the compound transposons does not depend on their size and takes place with the efficiency close to that characteristic of the intact Tn1000. Insertion of Tn5 into the gene coding for Tn1000 transposase results in sharp decrease in the efficiency of Tn1000 translocations. This effect, however, may be eliminated by introduction into the cell of the intact Tn1000.

Chromosome Mapping↗