Krishna K. Tewari

picture of Krishna K. Tewari

Professor, Molecular Biology and Biochemistry
School of Biological Sciences

PH.D., McGill University (Canada)

Phone: (949) 824-4738
Fax: (949) 824-8551

University of California, Irvine
1216 Natural Science I
Mail Code: 3900
Irvine, CA 92697
Research Interests
Chloroplast DNA Replication and Transcription
Research Abstract
Professor Tewari's laboratory is investigating the structural organization of genes in chloroplast DNA (ctDNA), replication of ctDNA, and transcription of ctDNA genes. Chloroplast DNA of higher plants exists as homogeneous double--stranded, closed-circular DNA molecules of 120-160 kpb. The ctDNA codes for its own ribosomal RNA, tRNAs and about 60-80 proteins.

Current experiments are focused on the mechanism of transcription and replication of ctDNA in vitro. RNA polymerase from pea chloroplasts have been highly purified through DEAE cellulose, heparin sepharose, phosphocellulose, and FPLC chromatography. The enzyme shows specificity to ctDNA templates and can synthesize rRNA, mRNA, and tRNA genes. Experiments are in progress to identify promoter sequences that are required for the recognition by the RNA polymerase for all three types of RNA chains. These experiments are being carried out by constructing deletion mutants of the sequences that surround transcription initiation sites of genes. The RNA polymerase contains about 12 polypeptides. Polyclonal and monoclonal antibodies are being obtained to study the question of multiple RNA polymerase(s) and identify polypeptides that are involved in initiation, elongation and termination of transcripts. Genes coding for the polypeptides of RNA polymerase also are being studied.

The replication of ctDNA is being studied by electron microscopy, DNA sequence analysis, and the use of purified enzymes. The locations of the two replication origins in pea ctDNA have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalan. There are two origins of replication separated from each other by about 6 kbp. A partially purified replication system has been obtained which faithfully replicates recombinants containing the origin of replication. Other regions of the pea ctDNA did not show any replication activity in vitro. A chloroplast specific DNA polymerase and a topoisomerase I have been purified to homogeneity. A DNA primase has also been identified. The replication of ctDNA is now being studied using these purified proteins. Genes coding for these proteins also are being investigated.

The laboratory also is studying in great detail the replication of ctDNA, which proceeds by the D-loop mechanism and involves both Cairn's replicative forms and intermediates produced by the rolling circle mechanism. The first phase of research concentrates on initiation of ctDNA replication and elongation of the newly synthesized DNA chains. The precise position of the D-loop replication origins is being determined by in vitro 5' end labeling using polynucleotide kinase and gamma-labeled dTTP, purifying the labeled D-loops in denaturing gels, hybridizing the D-loops with an appropriate restriction fragment of ctDNA, singlestrand nuclease digestion, and analysis in comparison to the DNA sequencing ladder.
McKown, R. and K.K. Tewari (1984). Purification and Properties of a DNA Polymerase from Pea Chloroplasts. Proc. Natl. Acad. Sci. 81:2354.
Sun, E., D.R. Shapiro, B.W Wu, and K.K. Tewari (1986). Specific in vifro Transcription of 16S rRNA Gene by Pea Chloroplast RNA Polymerase. Plant Mol. Biol. 6:429.
Gold, B., N. Carrillo, K.K. Tewari, and L. Bogorad (1987). Nucleotide Sequence of a Preferred Maize Chloroplast Genome Template for in vitro DNA Synthesis. Proc. Natl. Acad. Sci. 84:194.
Meeker, R., B. Nielsen, and K.K. Tewari (1988). Localization of Replication Origins in Pea Chloroplast DNA. Mol. Cell. Biol. 8:1216.
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