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[SANET-MG] introns and gm crops



October 11, 2004
Prof. Joe Cummins
?Introns and transgenic crops?
        The genes of higher plants that code for proteins  are most frequently made up
of  DNA sequences that   are interrupted versions of the code for the protein.
The sequences called exons code for the protein sequence , they are interrupted
by  from one to many  sequences called introns. The introns  are spliced out of
the RNA transcribed from the gene as the RNA message leaves the nucleus. Having
introns and splicing introns is essential to maintain high levels of  RNA
message in the cytoplasm  for production of elevated protein content in the
plant cell. The main transgenes used in genetically modified (GM) crops are
derived from bacterial genes that are not interrupted and for that reason
(along with the codon preferences of  bacteria) are poorly transcribed and  any
resulting RNA messages  and protein are very limited in amount. The codon
preference  is  modified by making synthetic approximations of  the bacterial
transgene but  even though the approximation is necessary it is not sufficient
to insure high level protein production. The majority of bacterial transgenes
in crop plants have been altered by adding an intron to enhance protein
production from the gene. However, it has been found that the added intron does
not have to interrupt the protein code but can be added between the promoter
gene and the protein start codon. That displaced intron is spliced from the
messenger RNA and facilitates the movement of the RNA message from nucleus to
cytoplasm  resulting in much higher levels of protein production from the
transgene.  Enhanced gene expression in plants is described in United states
patent 5,859,347 (1). Intron insertions may be used to adjust transcription
activity. For example,  parthenocarpic  fruit is desirable because fruit set
does not depend upon pollination,  an active parthenocarpic  transgene
produced auxin hormone that activated  placenta, ovules and derived tissues of
fruits including eggplant and tomato.However, the tomato fruits were malformed
because an intron enhancer activated production of too much auxin hormone,.
When the original intron was replaced with a less active intron  the
parthonocarpic tomato produced in the modified  plant  was normal in
appearance, The inserted intron can be used to create a wide range of gene
expression in a plant (2). A natural  plant gene for phosphoglucose isomerase
has been found to bear  an intron  in the leader sequence between promoter and
translation start signal but similar arrangements have not been reported in
animals(3).
        The enhancement of transcription by intron  was first studied in rice by
inserting  an intron within the protein coding sequence near the translation
start in a gene of bacterial origin. The inserted intron, from a castor bean
catalase gene, enhanced transcription  80 to 90 fold over the gene lacking an
intron. Interestingly, tobacco was enhanced to a lesser degree than rice  when
the catalase inton was inserted Different introns  are differently active
depending on the plant transcription machinery (4). The intron mediated
enhancement of transcription  is dependent on the proximity of the activation
intron to the translation start signal, introns nearest the start function best
and the transcription enhacement declined with distance form the promoter and
translation start signal even though the introns were spliced with equal
activity at all of the positions in a gene (5). Introns placed near the
promoter and translation start  most effectively enhance messenger RNA
production.
        Split genes appear in all organisms with nuclei but lower eukaryotes have
mainly unsplit genes  with some split genes while more evolved muticellular
organisms  have mostly all split genes. Introns are believed to have provided
a means of shuffling exons  by homologous recombination  (in homologous
recombination greater recombination between genes is achieved by increasing
distance between genes  along a chromosome). Exons correspond to domains
(modular regions of  a specified function) in a protein, so proteins with novel
activity can be rapidly  generated by recombination and thus provide diversity
for evolution (6). While lower eukaryotes mainly have ?constitutive? introns
(introns simply spliced out of the RNA message), higher organisms  have evolved
?alternate? introns(introns that may or may not be spliced) that allow
different proteins to be transcribed from the same gene. Genes of higher
organisms are frequently  made up from  a mixture of alternate and constitutive
 introns. Alternate introns may  either skip exons or the introns may be
retained and  read through during translation to create an additional domain.
Alternative introns are believed to have  evolved from mutations in the splice
sites of constitutive introns, those mutations   yield the benefit of diversity
for natural selection (7). The term ?trascriptosome? has been coined to deal
with an intra-nuclear  organelle that performs transcription and splicing
functions (8). Regulation of alternative splicing has been studied  in
Arabidopsis and rice along with  man, mouse and fruit flies. Regulatory
sequences are recognized on exons along with weak splice sites on alternative
introns  (9).
        A data base and analyses of alternatively  introns has been reported, the data
base shows that  the number of alternatively spliced genes in plants appears to
be much smaller than those in animals but the study of alternatively spliced
genes in plants has had much less investigation than that in animals and the
proportion of alternatively spliced genes in plants may increase as study
continues (10). Genes governing alternate splicing  from maize are similar to
splicing control genes of other organisms , splicing factors affect splice site
utilization  and the transcripts of maize splicing factors  are themselves
alternatively spliced  (11). The proportion of retained introns  (above 30%)
in arabidopsis was about three times higher than the proportion of retained
introns in humans (12). Tissue specific  alternate splicing was observed in
rice (13) and is presumably a key aspect of  alternate splicing in most plants.
Alternative splicing is clearly important in plants but the extent of its
function in plants is not yet clear.
        Introns are inserted  between the promoter and the translation start codon in
nearly all of  the commercial GM crops. These introns are needed to enhance
messenger RNA production and transfer to the cytoplasm of  synthetic genes
lacking introns derived from bacteria.  The peculiar location of the intron
sequence is not commonplace in plants but has been observed in natural plants.
Those regulating release of GM crops to the environment as food and feed  have
simply noted that introns have been included in the genetic modification. The
novelty of the intron insertion site is not discussed nor is any potential
alternative splicing of the synthetic transgenes.  Certainly, alternative
splicing should have been alluded to in the regulatory dossiers  but the
regulators seem to have ignored the process and its ramification  in production
of  novel proteins  that deserve regulatory scrutiny. In conclusion, regulators
 who approve GM crops for environment, food and feed  should add alternative
splicing to their vocabulary of concern.
References
1.      Brown,S. and Santino,C. Enhanced expression in plants 1999 United States
patent 5,859,347 pp1-31
2.      Pandolfini,T,Rotino,G, Camerini,S, Defez,R.and Spena,A. Optimisation of
transgene action at the post-transcriptional level:high quality parthenocarpic
fruits in industrial tomatoes  2002  BMC Biotechnology 2, 1-11
3.      Gottlieb L. and Ford, V. The 5' leader of plant PgiC has an intron: the
leader shows both the loss and maintenance of constraints compared with introns
and exons in the coding region.2002 Mol Biol Evol. 2002 19,1613-23
4.      Tanaka,A, Mita,S,Ohta,S,Kyozuka,J,Shimamoto,K. and  Nakamura,K. Enhancement
of foreign gene expression by a dicot intron in rice but not in tobacco is
correlated with an increased level of mRNA and an efficient splicing of the
intron  1990 Nucleic Acids Res. 18, 6767-70
5.      Rose,A. The effect of intron location on intron-mediated enhancement of gene
expression in Arabidopsis 2004 in press  doi: 10.1111/j.1365-313X.2004.02247.x
6.      Liu ,M.and Grigoriev,A. Protein domains correlate strongly with exons in
multiple eukaryotic genomes ? evidence of exon shuffling?  2004 Tends in
Genetics 20 No. 399-405
7.      Ast,G. How did alternative splicing evolve 2004 Nature Review Genetics
5,773-82
8.      Gall,J.,Bellini,M, Wu,Z. and Murphy,C. Assembly of the nuclear transcription
and processing machinery:Cajal bodies (coiled bodies) and transcriptosomes 1999
Mol.Bio.Cell,10,4385-4402
9.      Itoh,H,Washio,T. and Tomita,M. Computational comparative analyses of
alternate splicing regulation using full-length cDNA of various eukaryotes
2004RNA 10,1005-18
10.     Zhoua,Y,Zhoua,C,Yea,L,Donga,J,Xua,H,Caia,L,Zhanga,L. and  Wei ,L.  Database
and analyses of known alternatively spliced genes in plants 2003 Genomics  6,
584-95
11.     Gao,H,Gordon-Kamm,W. and  Lyznik,L. ASF/SF2-like maize pre-mRNA
splicing factors affect splice site utilization and their transcripts are
alternatively spliced    2004 Gene  339,25-37
12.     Ner-Gaon1,H,Halachmi1,R,Savaldi-Goldstein1,S, Rubin,E,  Ophir,R. and
Fluhr,R.  Intron retention is a major phenomenon in alternative splicing in
Arabidopsis 2004 The Plant Journal 39,877-85
13.     Isshiki,M,Nakajima,M,Satoh,H. And Shimamoto,K. dull:rice mutants with tissue
specific effects on the splicing of the waxy pre-mRNA 2000 The Plant Journal
23,451-60

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