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  • 1
    Electronic Resource
    Electronic Resource
    Nascent transcript-binding protein of the pea chloroplast transcriptionally active chromosome (1993)
    Springer
    Plant molecular biology 23 (1993), S. 963-979 
    Details
    ISSN: 1573-5028
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract This study describes the nascent RNA-binding protein of the pea chloroplast transcriptional complex. The protein has been identified by photoaffinity labelling of the transcriptionally active chromosome (TAC) which utilizes the endogenous plastid DNA as template. UV irradiation of lysed chloroplast or the isolated TAC under conditions optimized for transcription photocross-links nascent radiolabelled transcripts (up to 250 nucleotides in length) to a 48 kDa protein. The photoaffinity labelling of the transcript-binding protein is dependent on UV irradiation, is maximal after about 30 min of irradiation, and is completely dependent on transcriptional activity; no cross-linkage has been observed with presynthesized RNA. Cross-linkage is influenced by salts and inhibitors in accordance with their effects on transcription. The photoconjugate is composed of protein and RNA moieties, and can be hydrolysed by several proteases. However, the cross-linked transcript is protected from nucleases until the protein is removed. Manganese enhances photoaffinity labelling of the transcript-binding protein, and this is paralleled by an increase in total transcriptional activity of TAC. This protein was isolated by 2-dimensional polyacrylamide gel eletrophoresis and the sequence of 15 amino acid residues at the amino terminus was determined. The nascent transcript-binding protein appears to be involved in the transcription of all three classes of chloroplast genes. We also found a polypeptide of identical molecular weight to get cross-linked to nascent transcripts in chloroplasts isolated from other legumes such as Cicer arietenum, Vigna radiata and Phaseolus vulgaris, and monocots like Zea mays, Oryza sativa and Pennisetum americanum.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00021812
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  • 2
    Electronic Resource
    Electronic Resource
    Cloning and characterisation of a gene encoding an antiviral protein from Clerodendrum aculeatum L. (1997)
    Springer
    Plant molecular biology 33 (1997), S. 745-751 
    Details
    ISSN: 1573-5028
    Keywords: cDNA ; Clerodendrum aculeatum systemic resistence inducing protein ; plant virus inhibitor ; ribosome inactivating proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005716103632
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  • 3
    Electronic Resource
    Electronic Resource
    Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation (1999)
    Springer
    Plant molecular biology 41 (1999), S. 125-137 
    Details
    ISSN: 1573-5028
    Keywords: cell proliferation ; in vitro transcription and translation ; Pisum sativum ; plant promoter ; primer extension ; RACE-PCR ; transcript analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3′ end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5′-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006352820788
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  • 4
    Electronic Resource
    Electronic Resource
    Cloning, expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea (1998)
    Springer
    Plant molecular biology 37 (1998), S. 773-784 
    Details
    ISSN: 1573-5028
    Keywords: heterologous expression ; introduction of positive supercoils in DNA ; Pisum sativum ; RACE-PCR ; relaxation of DNA supercoils
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006086311875
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  • 5
    Electronic Resource
    Electronic Resource
    Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA (1986)
    Springer
    Plant molecular biology 6 (1986), S. 1-12 
    Details
    ISSN: 1573-5028
    Keywords: chloroplasts ; gene localization ; pea ; sequences ; tRNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Eight 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00021301
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  • 6
    Electronic Resource
    Electronic Resource
    Transformation of Nicotiana tabacum with a native cry1Ia5 gene confers complete protection against Heliothis armigera (1998)
    Springer
    Molecular breeding 4 (1998), S. 473-478 
    Details
    ISSN: 1572-9788
    Keywords: Bt genes ; transformation ; protection against insects ; cry1Ia5
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009696027993
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