[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]
[SANET-MG] background on GM chlamydomonas
June 24, 2005
Prof. Joe Cummins
“Producing human pharmaceutical proteins in a green alga, Chlamydomonas
reinhardtii”
Two proposals have recently been made to produce a number of
pharmaceutical proteins using a green alga, Chlamydomonas in the state
of Hawaii. Both of those proposals were couched in broad general terms
and made scant effort to critically evaluate the impact of the
recombinant algae on humans and the environment (1). The production of
human pharmaceutical proteins has been reported in journals but to
appreciate the actual process it is necessary to consider the basics of
the alga and its molecular genetic makeup. Chlamydomonas has three
genomes, nuclear, chloroplast and mitchondria. The nuclear genome has an
unusually high GC content (62%) with a DNA size about 100 mega base
pair. The high GC content of the nuclear genome creates a codin bias
that makes it difficult for foreign genes to be expressed until their
codons are adjusted. There are 17 nuclear chromosomes. There are two
mating types (+ the maternal gamete and – the paternal gamete). The
chloroplast genome consists of a circular DNA molecule of about 200
kilo base pairs (2). Prior to zygote formation the chloroplast DNA of
the + (maternal) gamete is methylated to protect it from degradation,
the -(paternal) DNA is degraded , thus chloroplast inheritance is
maternal(3). The chloroplast DNA has a GC content 34%. Foreign genes
more readily transform the chloroplast than the nuclear genome but in
many instances codon bias adjustments are needed(2).The Chlamydomonas
mitochondrial genome is tiny linear 15.8 kilobase DNA molecule the
mitochondrial ribosome genes are peculiar in that they synthesized as
fragments whose transcripts are joined to form a larger more typical
ribosomal RNA. The mitochondrial genome is transformed with great
difficulty. Finally, peculiarly, the mitochondrial genes are inherited
paternally, through the minus mating type (2). The Chlamydomonas
chloroplast has proved to be the most effective system for expressing
foreign transgenes, however, the transformation of the chloroplast
chromosome fundamentally differs from the transformation of nuclear
genes of algae and higher plants.
Transformation of nuclear genes in algae and higher plants is
achieved by illegitimate recombination involving DNA double strand
breaks and insertion of the transgenes at the the break site.
Chloroplast transformation resembles transformation in bacteria or
yeast , it is achieved using homologous ( legitimate) recombination.
Such transformations requires sequence tails at the ends of the gene to
be inserted, such sequence tails are homologous to a chloroplast
gene(4). Insertion of the transgene leads disruption of the gene into
which the transgene has been inserted. The transgene inserted is
targeted to particular gene so that it location is established at a
defined insertion site. The inserted transgene includes an appropriate
promoter and sequences at both the 5’ and 3’ untranslated regions of
the the transgenes. Such sequences govern RNA editing in which some
messenger RNA cytosine molecules are converted to uridine. RNA editing
is commonplace in chloroplasts and must be accounted for(5). It has been
noted that RNA editing sites appear more readily in genes (such as the
transgene), whose loss can be tolerated (6). Along with RNA editing some
chloroplast genes contain introns and some of those may have alternate
splicing such as has been observed in a chloroplast heat shock protein (7).
An example of the use of Chlamydomonas chloroplast to produce a
mammalian vaccine is the production for a foot and mouth disease
vaccine protein fused with a cholera toxin B subunit. The cholera toxin
B subunit a mucosal adjuvant that binds to intestinal epithelial
surfaces. The foot and mouth disease antigen gene fused to the
choleraB subunit gene was driven by the constitutive ATP stynthetase
(atpA) promoter and transcription was terminated by the rubisco (rbcL)
terminator. A spectomycin selective marker (aadA) was also inserted
downstream from the antigen-toxin fusion protein gene. At the 5’ (start)
end of the gene insert there was a short sequence from the
Chlamydomonas chloroplast chlorophyl L (chlL) and another short sequence
from the chlorophyll L (chlL) gene at the 3’ end of the insert, these
short sequences target the insert to the chloroplast chlL gene ,
disrupting its function. The disrupted strain had a “yellow in the dark”
phenotype in contrast to the “green in the dark” phenotype of the wild
type alga.The foot and mouth antigen-cholera toxin B fusion protein
produced in Chlamydomonas made up about 3% of the soluble protein in
the algal cell(8). The algal system was satisfactory for producing a
mucosal vaccine source.
The problems that may be encountered include the predictable release of
modified Chlamyomonas to the environment. Chlamydomonas is commonly
associated with the soil and aquatic environment and contamination of
the environment would lead to dispersal of the gene for spectinomycin
resistance , the antibiotic is currently employed in treating a number
of infections. Pollution of the aquatic and soil environment with the
modified alga would, also, create oral tolerance in farm animals leading
to tolerance to the pathogen rendering the vaccine ineffective. Finally,
the potential impact of RNA editing on the vaccine construction was not
discussed even though it is a potential source of dangerous variability
in a vaccine.
A good deal more thought should be given to the environmental hazards
associated with the production of recombinant algae bearing vaccines
and other pharmaceuticals.
References
1.Cummins,J. Algal recombinant pharmaceuticals in Hawaii 18 June 2005
Brief to Hearing,Plant Quarentine Honolulu Office
2.Rochaix,J. The three genomes of Chlamydomonas Photosynthesis Research
2002,73,285-93
3. Nishiyama R, Wada Y, Mibu M, Yamaguchi Y, Shimogawara K and Sano H.
Role of a nonselective de novo DNA methyltransferase in maternal
inheritance of chloroplast genes in the green alga, Chlamydomonas
reinhardtii Genetics. 2004 Oct;168(2):809-16
4. Staub,J. Expression of eukaryotic peptides in plant plastids United
States Patent 6,812,379 2004
5. Hefitietz,P. and Tuttle,A. Protein expression in plastids Current
Opinion in Plant Biology 2001,4,157-61
6. Fiebig A, Stegemann S and Bock R. Rapid evolution of RNA editing
sites in a small non-essential plastid gene Nucleic Acids Res. 2004 Jul
7;32(12):3615-22
7. Schroda M, Vallon O, Whitelegge JP, Beck CF and Wollman FA. The
chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by
differential splicing. Plant Cell. 2001 Dec;13(12):2823-39
8. Sun M, Qian K, Su N, Chang H, Liu J and Shen G. Foot-and-mouth
disease virus VP1 protein fused with cholera toxin B subunit expressed
in Chlamydomonas reinhardtii chloroplast. Biotechnol Lett. 2003
Jul;25(13):1087-92.
********************************************************
To unsubscribe from SANET-MG:
1- Visit http://lists.sare.org/archives/sanet-mg.html to unsubscribe or;
2- Send a message to <listserv@sare.org> from the address subscribed to the list. Type "unsubscribe sanet-mg" in the body of the message.
Visit the SANET-MG archives at: http://lists.sare.org/archives/sanet-mg.html
For more information on grants and other resources available through the SARE program, please visit http://www.sare.org.