Krishna K. Tewari

Pea chloroplast topoisomerase I: purification, characterization, and role in replication

A DNA-relaxing enzyme was purified 5 000-fold to homogeneity from isolated chloroplasts of Pisum sativum. The enzyme consists of a single polypeptide of 112 kDa. The enzyme was able to relax negatively supercoiled DNA in the absence of ATP. It is resistant to nalidixic acid and novobiocin, and causes a unit change in the linkage number of supercoiled DNA. The enzyme shows optimum activity at 37°C with 50 mM KCl and 10 mM MgCl2. From these properties, the enzyme can be classified as a prokaryotic type I topoisomerase.Using a partiall purified pea chloroplast DNA polymerase fraction devoid of topoisomerase I activity for in vitro replication on clones containing the pea chloroplast DNA origins of replication, a 2–6-fold stimulation of replication activity was obtained when the purified topoisomerase I was added to the reaction at 70–100 mM KCl. However, when the same reaction was carried out at 125 mM KCl, which does not affect DNA polymerase activity on calf thymus DNA but is completely inhibitory for topoisomerase I activity, a 4-fold drop in activity resulted. Novobiocin, an inhibitor of topoisomerase II, was not found to inhibit the in vitro replication of chloroplast DNA.

Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA

SummaryEight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNAVal(GAC), tRNAAsn(GUU), tRNAArg(ACG), tRNALeu(CAA), tRNATyr(GUA), tRNAGlu(UUC), tRNAHis(GUG), and tRNAArg(UCU) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition,two other tRNA genes, tRNAGly (UCC) and tRNAIle(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90–100% homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5′ flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNAGly(UCC), and the tRNAGlu(UUC) contains GATTC in its T-loop.

Specific in vitro transcription of 16S rRNA gene by pea chloroplast RNA polymerase

SummaryA highly purified RNA polymerase preparation from pea chloroplasts has been shown to specifically transcribe the 16S rRNA gene in vitro using the recombinant pCB2-8 DNA as a template. The RNA polymerase has been found to show maximum activity and specificity with pea supercoiled rDNA as a template. At low concentrations of ribonucleoside triphosphates, the RNA polymerase selectively initiates transcription on the 16S rRNA gene. A part of the 16S rRNA gene has been sequenced. The mature 16S rRNA has been found by S1 nuclease analysis to contain sequences starting from GAAGCT. The in vitro synthesized RNA has been found to protect the same nucleotides GAAGCT. In addition, the in vitro synthesized RNA was also found to strongly protect bases starting with TATG located at about 260 bases away from the start site of the mature 16S rRNA.

CLONING, EXPRESSION AND CHARACTERIZATION OF A GENE WHICH ENCODES A TOPOISOMERASE I WITH POSITIVE SUPERCOILING ACTIVITY IN PEA

We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5′ end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg2+. In the presence of Mg2+ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.

Transformation of Nicotiana tabacum with a native cry1Ia5 gene confers complete protection against Heliothis armigera

A cry1Ia5 insecticidal toxin coding gene has been cloned from an Indian isolate of Bacillus thuringiensis. Sequence analyses of the cry1Ia5 gene revealed the absence of potential polyadenylation signal sequences thus making it a suitable candidate for expression in plants without extensive modification. This possibility was examined by subcloning the cry1Ia5 gene into a plant expression vector and then transferring it to Nicotiana tabacum through Agrobacterium-mediated transformation. Our results demonstrate that N. tabacum with a stably integrated native cry1Ia5 gene afforded complete protection against predation by Heliothis armigera. Forty three percent of the transgenic plants displayed a high level of protection against insect predation. The protection obtained in transgenic plants with the cry1Ia5 gene was comparable to that obtained with the synthetically modified cry1A(b) or cry1A(c) genes. The results demonstrate that novel insecticidal genes already exist in nature that do not require extensive modifications for efficient expression in plants.

Pea chloroplast DNA primase: characterization and role in initiation of replication

AbstractA DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.

CLONING AND CHARACTERISATION OF A GENE ENCODING AN ANTIVIRAL PROTEIN FROM CLERODENDRUM ACULEATUM L.

The Clerodendrum aculeatum-systemic resistence inducing (CA-SRI) protein, a 34 kDa basic protein, plays a key role in inducing strong systemic resistance in susceptible plants against various plant viruses [22]. We have cloned the cDNA encoding the CA-SRI from C. aculeatum leaves using antibodies raised against the purified protein and degenerate oligonucleotide probes derived from microsequencing of the CA-SRI protein. The full-length cDNA consisted of 1218 nucleotides with an open reading frame of 906 bp. The deduced amino acid sequence of CA-SRI protein showed varying homology (ranging from 11 to 54%) to the ribosome inactivating proteins (RIPs) from other plant species. CA-SRI inhibited in vitro protein synthesis both in rabbit reticulocyte lysate and wheat germ lysate but not in Escherichia coli in vitro translation system. The CA-SRI open reading frame was expressed in an E. coli expression vector and the purified recombinant protein inhibited protein synthesis in rabbit reticulocyte lysate. Southern blot analysis indicated that the CA-SRI gene may be present in low copy number.

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA.

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3′ end using tRNA nucleotidyl transferase and α32P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.

Structure of Chloroplast DNA

We had previously reported the isolation of circular chloro-plast (et) DNA molecules from pea leaves. Circular pea ctDNA was found to have a molecular weight of 90×106 with no evidence of inter- or intramolecular heterogeneity. Recently we have extensively studied the size and structure of ctDNAs from pea, bean, spinach, lettuce, corn, and oats.2 As much as 89% of the ctDNAs from these higher plants has been obtained in circular form. The DNA preparations were also found to contain circular and catenated dimers of the circular monomer. The molecular sizes of these circular ctDNA molecules relative to internal standards has been found to range from 85×106 to 97×106. The molecular size of these ctDNAs was also determined by renaturation kinetics and found to range from 82x106 to 93×106. The excellent agreement between the molecular weights of these circular DNA molecules obtained by electron microscopy and the molecular weight of the unique sequences of these ctDNAs determined by renaturation kinetics suggests that the sequence of a circular ctDNA molecule represents the entire information content of the ctDNA.

Analyses of the extent of immunological relatedness between a highly purified pea chloroplast functional RNA polymerase and Escherichia coli RNA polymerase

Summary The reported nucleotide sequence homology between the RNA polymerase (EC 2.7.7.6) coding (rpo) genes in Escherichia coli and their putative homologues in chloroplast DNA is not only limited to about 26-50 %, but also scattered. Utilising the homologous in vitro transcription system and immunological approach, we address as to which extent the chloroplast functional RNA polymerase and E. coli RNA polymerase are related. Highly purified RNA polymerase from pea chloroplasts has been demonstrated to transcribe both ribosomal and messenger RNA genes and the purified homologous antibodies inhibit the chloroplast gene promoter recognizing ability as well as in vitro transcriptional activity of the enzyme [Rajasekhar et al., 1991]. While recognizing all of the polypeptides of the chloroplast enzyme on Western blots, the chloroplast RNA polymerase antibodies did not cross-react with the sub-units of E. coli RNA polymerase or wheat germ RNA polymerase and vice versa. Antibodies against the 130 kDa,110 kDa, 75-95 kDa and 48 kDa polypeptides of the purified chloroplast RNA polymerase immobilized only homologous RNA polymerase on the affinity columns. The active enzyme eluted from the immunoaffinity columns showed sub-unit patterns similar to that of purified RNA polymerase. The specificity of these antibodies was confirmed with the lack of immobilization of E. coli RNA polymerase and vice versa with the E. coli RNA polymerase antibodies. The E. coli RNA polymerase also manifested transcription in vitro utilising the supercoiled 7A/30T recombinant containing the chloroplast 16S rRNA minigene as well as the supercoiled pCB 1-3 recombinant containing the upstream sequences of ribulose-1,5-bisphosphate carboxylase large sub-unit (rbc-L) gene. The transcriptional activities, however, were specifically affected only by homologous RNA polymerase antibodies, but not by heterologous RNA polymerase antibodies. While both the chloroplast and the E. coli RNA polymerases caused mobility shifts of the chloroplast 16S rRNA gene and rbc-L gene promoter fragments, only the homologous antibodies were effective in abolishing this response. The data obtained form the first functional evidence that the highly purified pea chloroplast RNA polymerase and the E. coli RNA polymerase are immunologically only distantly related.