Barnase is toxic to humans and other mammals！ http://www.ibiblio.org/ecolandtech/SoilWiki/message-archives/JoeCummins/msg00853.html *Barnase Ribonuclease is toxic to Humans and other Mammals* Barnase ribonuclease is an enzyme toxic to cells, it is produced in nature by the bacterium, Bacillus amyloliquefaciens, which synthesizes and secretes the enzyme . The enzyme is not toxic to the bacterium that produces it because it is inhibited by another protein, barstar, produced within that bacterium. As the enzyme is secreted from the bacterial cell barstar is removed from its complex with barstar. The bacterial gene for barnase and the gene for barstar have been employed extensively in transgenic plants and animals to set up regulatory systems that are capable of ablating (killing) specific organs or tissues of the organism by the interplay of barnase and barstar. Mustard (Brassica Juncea) was modified with the barnase gene driven by a pollen specific (tapetum) promoter that produced male sterile lines of the mustard plants by ablating pollen cells (1) Male sterile plants can be used to limit pollen release from a crop or to produce hybrids. United States Patent 6,509,516 deals with producing male sterile Brassica employing barnase (2), while United States patent 6,969.786 deals with producing hybrid canola (Brassica napa or B.napus) using a male sterile line created using barnase and barstar genes (3).The production of Brassica hybrids using the barnase-barstar system has been illustrated and explained (4). Barnase, unaccompanied by its specific inhibitor barstar, is known to be a potent cell poison . Traces of barnase are toxic to the rat kidney and to human cell lines . Barnase is actually being exploited as a conditional ‘suicide gene’ to cause celldeath in mammalian and human cells when it is induced. Cell toxicity caused by barnase may be affected by RNA interference (RNAi) (10).The hazard associated with RNAi is shown by experiments over saturating small RNA pathways leading to the death of many experimental mice (11).RNAi is known to circulate among the cells of plants (12) so the ablation of anther cells may be accompanied by RNAi spread throughout the entire plant. The pollen produced by the hybrid mustard actually contains the barnase gene combined with the barstar. Is barnase expressed at low basal levels in the plant tissues when it is not in the induced state (is the regulatory transgene leaky)? Are the constructs sufficiently stable to ensure that the barnase is only active in the anther? How much RNAi is translocated throughout the plant?As indicated earlier, Barnase, even if expressed at low levels could prove toxic to a wide range of animals that interact with the plant, including not only humans, but also rodents and bees. It could also enter the human food chain in bee honey. As pointed out earlier, in bacteria, barnase is synthesized but immediately joined to barstar to avoid cell death. The newly synthesized barnase (combined with barstar) has a short leader sequence at the N-terminus, that sequence directs t the barnase –barstar complex to the cell membrane. As barnase is secreted from the cell membrane barstar is separated from barnase and left behind in the cell interior (13). If barnase can be separated from barstar at the cell membrane of bacteria there are likely to be situations where plant cells separate barnase from barstar. The pathway for folding and the stability of barnase has been studied extensively (14). It seems likely that the barstar-barnase complex is very stable and likely to persist in the grain of food crops such as mustard. However, in spite of the extensive use of the barnase-barstar systems in Brassica such as mustard and oilseed rape (canola), there does not seem to have been any effort to measure the quantity of the barnase-barstar complex in the grain of the food crops. The barnase-barstar complex in transgenic mustard is likely to be produce a strong immunological response such as inflammation or it may even be an allergen. However, these aspects to not appear to have been studied. There is evidence that barstar induces a strong immune response (15). However, the barnase-barstart complex needs fuller study in terms of immune responses, including allergy. The barnase-barstar system for pollen control has been used with both mustard and with canola. The canola label is used with two species Brassica rapa (Polish rapeseed) and Brassic napus (Argentine rapeseed) (16) Mustard (Brassica juncea) may produce inter specific hybrids with canola (B. rapa) (17) but such hybrids are not yet grown commercially. It is wisest to consider the hazards of barnase-barstar transgenic crops as being separate but related , not as being identical as has been done in some safety evaluations. In conclusion, the actual toxicity of barnase and the toxicity of the barnase-barstar complex in food and feed cries out for fuller study regarding the impact on humans and animals. The main areas requiring fuller study prior to the exposure of millions of people and millions of animals to the toxins are the exposure to the highly toxic barnase ribonuclease on consuming transgenic food and the potential toxic immune responses to the bartnase-barstar complex in the transgenic crops. References 1.Jagannath,A,Bandyopadhyay,P,Arumugam,N, Gupta,V,Burma,P and Pental,D. The use of a spacer DNA fragment insulates the tissue-specific expression of a cytotoxic gene (barnase) and allows high frequency generation of transgenic male sterile lines in Brassica juncea. Molecular Breeding 2001,8,11-21 2. Weston,B. and deBeuckeleer,M Male sterile brassica plants and methods for producing same 2003 US Patent 6,509,516 3.Grombacker,A and Patel,D. Canola line 43A56 2003 US Patent 6,969,786 4. Ho, M-W and Cummins,J. Chronicle of an ecological disaster foretold Isis Report 2003 http://www.isis.org.uk/ 5. Cummins J. Terminator gene product alert, ISIS News 6, September 2000, ISSN: 1474-1547 (print), ISSN: 1474-1814 (online). 6. Ilinskaya O and Vamvakas S. Nephrotic effect of bacterial ribonucleases in the isolated and perfused rat kidney. Toxicology 1997, 120, 55-63. 7. Prior T, Kunwar S and Pastan I. Studies on the activity of barnase toxins in vitro and in vivo. Biocong Chem 1996, 7,23-9. 8. Leuchtenberger S, Perz A, Gatz C and Bartsch JW. Conditional cell ablation by stringent tetracycline-dependent regulation of barnase in mammalian cells. Nucleic Acids Research 2001, 29 (16). 9. Bi YM, Rothstein SJ and Wildeman AG. A novel strategy for regulated expression of a cytotoxic gene. Gene 2001, 279, 175-9. 10.Ilinskaya,O and Makarov,A. Why ribonucleases induce tumor cell death Molecular Biology 2005,39,3-13 11. Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR, Marion P, Salazar F and Kay MA. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature. 2006 May 25;441(7092):537-41 12. Tournier B, Tabler M and Kalantidis K. Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants. Plant J. 2006 Aug;47(3):383-94 13. Paddon CJ, Vasantha N and Hartley RW. Translation and processing of Bacillus amyloliquefaciens extracellular RNase. J Bacteriol. 1989 Feb;171(2):1185-7 14. Fersht AR. The sixth Datta Lecture. Protein folding and stability: the pathway of folding of barnase. FEBS Lett. 1993 Jun 28;325(1-2):5-16 15.Kirkham,P,Nori,D and Winter,G. Towards the design of an antibody that recognizes a given protein epitope J. Mol. Biol. 1999,285,909-15 16. Canola Council of Canada Origin and History of Canola 2006 http://www.canola-council.org/PDF/canola/english/originhistory.pdf#zoom=100 17. Choudhary,R,Joshi,P and Rao,R. Cytogenetics of Brassica junceaXBrassica rapa hybrids and patterns of variation in the hybrid derivatives Plant Breeding 2002 ,121,292-6
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