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[SANET-MG] barnase is toxic to humans and other mammals
*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
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.
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
vitro and in vivo. Biocong Chem 1996, 7,23-9.
8. Leuchtenberger S, Perz A, Gatz C and Bartsch JW. Conditional cell
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
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
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
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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