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1.
Plant Physiol ; 124(3): 927-34, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11080270

ABSTRACT

Guttation, the loss of water and dissolved materials from uninjured plant organs, is a common phenomenon in higher plants. By using endoplasmic reticulum signal peptides fused to the recombinant protein sequences, we have generated transgenic tobacco (Nicotiana tabacum L. cv Wisconsin) plants that secrete three heterologous proteins of different genetic backgrounds (bacterial xylanase, green fluorescent protein of jellyfish [Aequorea victoria], and human placental alkaline phosphatase) through the leaf intercellular space into tobacco guttation fluid. Production rates of 1.1 microg/g of leaf dry weight per day were achieved for alkaline phosphatase with this protein comprising almost 3% of total soluble protein in the guttation fluid. Guttation fluid can be collected throughout a plant's life, thus providing a continuous and nondestructive system for recombinant protein production. Guttation fluid has the potential of increasing the efficiency of recombinant protein production technology by increasing yield, abolishing extraction, and simplifying its downstream processing.


Subject(s)
Alkaline Phosphatase/biosynthesis , Luminescent Proteins/biosynthesis , Nicotiana/genetics , Plants, Toxic , Recombinant Fusion Proteins/biosynthesis , Xylosidases/biosynthesis , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Clostridium/genetics , Enzyme-Linked Immunosorbent Assay , Green Fluorescent Proteins , Humans , Luminescent Measurements , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Plant Leaves/metabolism , Plant Leaves/physiology , Plant Leaves/ultrastructure , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/physiology , Recombinant Fusion Proteins/metabolism , Nicotiana/metabolism , Nicotiana/physiology , Xylan Endo-1,3-beta-Xylosidase , Xylosidases/genetics , Xylosidases/metabolism
2.
Mol Biol Evol ; 16(3): 311-20, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10331258

ABSTRACT

Origin and rearrangement of ribosomal DNA repeats in natural allotetraploid Nicotiana tabacum are described. Comparative sequence analysis of the intergenic spacer (IGS) regions of Nicotiana tomentosiformis (the paternal diploid progenitor) and Nicotiana sylvestris (the maternal diploid progenitor) showed species-specific molecular features. These markers allowed us to trace the molecular evolution of parental rDNA in the allopolyploid genome of N. tabacum; at least the majority of tobacco rDNA repeats originated from N. tomentosiformis, which endured reconstruction of subrepeated regions in the IGS. We infer that after hybridization of the parental diploid species, rDNA with a longer IGS, donated by N. tomentosiformis, dominated over the rDNA with a shorter IGS from N. sylvestris; the latter was then eliminated from the allopolyploid genome. Thus, repeated sequences in allopolyploid genomes are targets for molecular rearrangement, demonstrating the dynamic nature of allopolyploid genomes.


Subject(s)
DNA, Plant/genetics , DNA, Ribosomal/genetics , Nicotiana/genetics , Plants, Toxic , Base Sequence , Evolution, Molecular , Gene Rearrangement , Genome, Plant , Molecular Sequence Data , Polymorphism, Genetic , Polyploidy , Repetitive Sequences, Nucleic Acid , Restriction Mapping , Sequence Homology, Nucleic Acid , Species Specificity
3.
Nat Biotechnol ; 17(5): 466-9, 1999 May.
Article in English | MEDLINE | ID: mdl-10331806

ABSTRACT

The large-scale production of recombinant proteins in plants is limited by relatively low yields and difficulties in extraction and purification. These problems were addressed by engineering tobacco plants to continuously secrete recombinant proteins from their roots into a simple hydroponic medium. Three heterologous proteins of diverse origins (green fluorescent protein of jellyfish, human placental alkaline phosphatase [SEAP], and bacterial xylanase) were produced using the root secretion method (rhizosecretion). Protein secretion was dependent on the presence of the endoplasmic reticulum signal peptide fused to the recombinant protein sequence. All three secreted proteins retained their biological activity and, as shown for SEAP, accumulated in much higher amounts in the medium than in the root tissue.


Subject(s)
Cloning, Molecular/methods , Nicotiana/genetics , Plant Roots/metabolism , Plants, Toxic , Recombinant Proteins/genetics , Alkaline Phosphatase/genetics , Alkaline Phosphatase/metabolism , Animals , Clostridium botulinum/enzymology , Clostridium botulinum/genetics , Green Fluorescent Proteins , Humans , Hydroponics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Placenta/enzymology , Plants, Genetically Modified , Recombinant Proteins/metabolism , Scyphozoa/genetics , Scyphozoa/metabolism , Nicotiana/growth & development , Nicotiana/metabolism , Xylan Endo-1,3-beta-Xylosidase , Xylosidases/genetics , Xylosidases/metabolism
4.
Proc Natl Acad Sci U S A ; 96(11): 5973-7, 1999 May 25.
Article in English | MEDLINE | ID: mdl-10339526

ABSTRACT

Alternative agriculture, which expands the uses of plants well beyond food and fiber, is beginning to change plant biology. Two plant-based biotechnologies were recently developed that take advantage of the ability of plant roots to absorb or secrete various substances. They are (i) phytoextraction, the use of plants to remove pollutants from the environment and (ii) rhizosecretion, a subset of molecular farming, designed to produce and secrete valuable natural products and recombinant proteins from roots. Here we discuss recent advances in these technologies and assess their potential in soil remediation, drug discovery, and molecular farming.


Subject(s)
Agriculture/methods , Biotechnology/methods , Soil Pollutants , Soil , Animals , Plant Roots , Plants, Genetically Modified , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism
5.
Plant Mol Biol ; 35(5): 655-60, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9349286

ABSTRACT

The nucleotide sequence of the intergenic spacer (IGS) region between the 25S and the 18S rRNA coding regions has been determined for tobacco (Nicotiana tabacum). The IGS (5140 bp in length) can be subdivided into several regions (I-VII) two of which, upstream and downstream of the putative transcription initiation site (TIS), contain prominent subrepeats (A and C). The unique sequence in the central part of the IGS (region IV) preceding the TIS is extremely AT-rich. The distance from the putative TIS to the 5' end of the 18S rRNA gene is 3005 bp. The IGS sequences are compared with potato (Solanum tuberosum) and tomato (Lycopersicon esculentum) IGS. Restriction mapping of 13 Nicotiana species shows that considerable rDNA repeat length heterogeneity in this genus is probably due to different numbers of A and C subrepeats.


Subject(s)
DNA, Plant/genetics , DNA, Ribosomal/genetics , Nicotiana/genetics , Plants, Toxic , Base Composition , Base Sequence/genetics , DNA, Plant/chemistry , DNA, Ribosomal/chemistry , Genetic Variation/genetics , Molecular Sequence Data , RNA, Ribosomal/genetics , RNA, Ribosomal, 18S/genetics , Restriction Mapping
6.
Theor Appl Genet ; 76(1): 108-12, 1988 Jul.
Article in English | MEDLINE | ID: mdl-24231990

ABSTRACT

Behavior of ribosomal RNA genes in the process of somatic hybridization was analyzed using hybrids Nicotiana tabacum + Atropa belladonna. Blothybridization of parental species DNAs to (32)P-rDNA specific probes revealed two classes of ribosomal repeats in both tobacco and nightshade; their length was 11.2 kb, 10.4 kb (tobacco) and 9.4 kb, 10.2 kb (night-shade). For analysis of hybrids, labelled (32)P rDNA specific probes were hybridized to DNA of parental species and somatic hybrids digested with restriction endonucleases EcoR1, EcoRV and BamH1. A new class of ribosomal DNA repeat, absent in parental species, was found in hybrid line NtAb-1. Possible mechanisms of appearence of a new rDNA class in the process of somatic cell fusion are discussed.

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