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Date:

November 13, 2000

Subject:

Response from Mae-Wan Camp; GM and Natural Genetic

 

Dear Friends:

I recently forwarded some discussion we had recently on Mae-Wan Ho's
writings and views in this group to Dr. Mae-Wan Ho for her response. As
Mae-Wan is traveling, her colleague Angela Ryan sends the following
response with a commentary on genome plasticity and asks how the
transgenes would affect it. Please respond to this group as I would
forward the replies to Angela,

Prakash
-----------------------

From: Angela Ryan
Subject: From ISIS

Dear Prakash

Please!!! - how can we have an informed scientific debate with people who
do not understand how to conduct themselves. This is both necessary and
worth while. Mae-wan is away at the moment but quite honestly the
comments are very nasty indeed and do not deserve to be taken seriously,
in my view.

Aren't we meant to be discussing GMOs? And why do I find it so hard to
engage your side in debate of the real scientific problems of GM?

In an attempt to redeem this sorry situation between our respected
supporters and enhance the GM debate, I attached a discussion paper on
natural genetic engineering. Perhaps you would be so kind as to share it
for those scientists who genuinely care to have open dialog on this topic.

Also, I pose a question, for focus;
How may GM effect the molecular systems that perform natural genetic
engineering events?

I look forward to the replies. Thank you

Best wishes
Angela Ryan, Molecular Biologist, ISIS

----------------------
Discussion Paper
Natural Genetic Engineering - Directed Mutations?
Note taken from : ANNALS NEW YORK ACADEMY OF SCIENCES (May 1999) ; CHANCE
FAVORS THE PREPARED GENOME by Lynn Helena Caporale

Compiled by Angela Ryan ISIS

Genomic mechanisms that adapt pre-existing functional DNA to new purposes
have been highly conserved in nature. The probability of some mutations
is so high that they can be easily predicted through the location of the
leading or lagging strand e.g. to form quasipalindromic secondary
structures that often mutate or ‘correct’ to exact palindromes, allowing
DNA repeats to change in length.

Horizontal transfer of genes and gene clusters between organisms is
common. Bacteria sample and adapt information available in their
environment – taking up and incorporating DNA and combining new properties
eg. Joining together of separately acquired catalytic and binding domains
of B1,4 endoglucanase. Intergrons are moving ‘cassettes’ that insert
antibiotic resistance, pathogenicity and other properties.

Exons and the sliding of the intron/exon boundary – sequences that enter
or leave a protein. Ie. Immunoglobulin hinge region, where variation in
protein structure can be accommodated. These changes may have larger
effects due to context effects leading to exposure of cryptic splice
sites, or even potential promoters/enhancers. Intron sequences may enter
exons through editing. Sequences in introns do facilitate duplication and
recombination of exons and effect the mutation rate of neighboring exons.

The efficiencies of duplication/variation have lead to large superfamilies
around useful scaffolds and active sites. Duplication has created
homologous regulatory pathways and cascades that, like duplicated genes,
can take on new roles by becoming connected to new upstream and or
downstream modules. Genomes have evolved molecular systems that have the
ability to mark duplicates for accelerated diversification and to protect
against the risk of massive loss of duplicated sequences by homologous
recombination.

"EVOLUTION IS TRANSLOCATION AND TRANSPOSITION, rather than point mutation.
Transposable elements have been instrumental in sculpting the
contemporary genome of all organisms"( Nina Fedoroff). Genetic repeats
and transposable elements "modularize" the genome by creating segments
that recombine and rearrange, playing an active role in genome
reorganisation - evolution. Modules are created by flanking DNA sequences
that increase the rate of recombination eg. Z-DNA promotes recombination
and is active at the boundary of insertion sites in heat shock protein
promoters in Xenpus, associated with class switch in immunoglobulins,
gene conversion in primates and multiple duplication and interchromosomal
transfer of gene-rich region. Low frequency recombination at widespread
sites that deviate from consensus sequences is very import for evolution
(Werner Arber) REPEATS create homology for recombination at non-allelic
sites eg. T-cell receptor, ALU sequences.

Transposable elements can translocate between, not within, functional
modules. Therefore repetitive sequences and transposable elements avoid
disruption of function. Although transposable element insertion does
inactivate genes, it is often reversible. It is thought that the invasion
of the genome by a transposable element has given rise to the emergence of
the vertebrate immune system. A transposon is proposed to have brought the
helix-turn helix DNA binding domains PAIRED box and POU, which are absent
in yeast, into an ancestral animal genome ( Susanna Lewis).

Recombination links each functional domain with other domains that may
affect its interaction with other proteins, regulate its synthesis and
activity, affect its half life, and or direct it to the nucleus,
cytoplasm, or cell membrane. "Thus ‘patchwork’ genes evolve…Just as
interchangeable parts enabled the industrial revolution!!!" (mechanistic
description fails) Functional regions of DNA ranging in size from gene
fragments to entire genomes appear to have been duplicated and/or combined
- entire genomes have joined together during evolution.

‘Genetic Exploration’ – ALU was identified at the site of recombination
responsible for a deletion in BRACA1 - increasing the risk of breast
cancer. Tandem repeat expansion, to risky recombination at ALU and L1,
represent ‘stumbling genomes’ that tolerate increased rates of variation
at such sites. ‘Genetic exploration’ forms the basis of new gene
families. The genome has learnt which connections are most likely to be
useful. Mobile elements insert into regions that regulate gene
expression. Original and duplicated genes both affect the expression of
neighbouring genes in response to conserved regulatory signals. Therefore
genes at both the original and new genomic locations may be turned on, or
repressed, together. Duplication and movement of regulatory regions can
link, through regulation , sets of loci that are not physically adjacent
in the genome. And when the regulatory region is within a mobile
element, expression can be unlinked by excision of the element.
Reversible inversions, which can subject genes to different regulatory
influences like an on-off switch ( Werner Arber) eg HOX GENES (or GOD
GENES) show over-riding evolutionary importance of alterations in
regulatory sequences, which explore body plans in physical spacetime
through efficient strategies that explore DNA sequence space (Frank
Ruddle) . Protein coding regions of certain hox genes can be exchanged
without effect and changes in the regulation of Hox gene expression play
a dominant role in evolution of body plans. Jim Shapiro – "the most
significant steps are often changes in the timing and location of a
protein’s synthesis, rather that changes in its amino acid sequence, which
demonstrate that a complex regulation system has evolved". Tandem
repeats are characterized as ‘tuning knobs’ because of their genetic
instability they represent a possible mechanism for fast multilocus
adaptation, dialing up and down the level of expression as the number of
repeats in a tandem array increases and decreases when the polymerase
slips. There may also be regulation of the likelihood that a slip will be
repaired – the tandem repeats we use as markers for quantitative traits
might BE quantitative traits.

DNA movement and DNA function – what’s the connection?

Shapiro emphasizes the genome’s capacity, in diverse phyla, for
‘coordinated multilocus changes’ and regulated genome rearrangements and
suggests several biochemical mechanisms that can connect mobile element
insertion with gene activity.

The extent that genetic variation responds to the environment?

Genomes have evolved the capacity to respond to predictable environment
challenges with predictable genetic changes e.g. the vertebrate immune
system and variation of surface antigens by pathogens seeking to hide from
the immune system. Environmental stress can alter the balance between
stability/repair and exploration. Under metabolic stress, organisms may
increase genetic variation and change the spectrum of mutations – Nina
Fedoroff encourages the study of genetic change at both local and global
levels – eg. Genetic changes that arise in episomes, and the uptake of DNA
from the environment can be increased during nutritional stress. Also the
induction of DNA uptake over sporulation, plus barriers to incorporation
of DNA may be relaxed, or movement of transposons can be held in check -
in a regulated, heritable, and reversible manner (methylation), then
released under stress. The potential when transposons are ‘released’ is
significant ie. a third of the human genome may be contained in transposon
DNA. Stress can increase variation in the absence of cell division, by
inducing double strand breaks: once the DNA is broken, recombination
‘repair’ can increase the frequency of nearby base substitution and
frameshifts. Recombination can increase variation not only by
recombination of existing alleles, but also by creating new ones’ (Foster)

RNA and RNA intermediates – editing, trans-splicing, and reverse
transcription
New sequences can be created and or explored in DNA, then pass into DNA
genomes through RT. Reverse transcripts and their chromosomal homologs
can recombine: thus retrotransposition and double strand breaks may play a
role in gene conversion. This can be regulated, because double strand
breaks can be regulated ( see Kenter)

FOCUSED, REGULATED, GENETIC VARIATION

The vertebrate immune system is a paradigm. Information in a gene that
accelerates binding site sequence exploration can be conserved when gene
family members duplicate, and thus become a feature of a gene family. E.g
genomic loci encoding heavy chains, light chains, and the T-cell receptor
all maintain mechanism for generation of focused diversity. Thus, gene
families should be analysed as systems rather that as lists of related
genes. In short the immune system provides striking evidence that genetic
exploration can be focused with a gene and can be regulated by the
environment e.g. newly evolved V regions.

Pathogens can also focus genetic variation, often changing surface
proteins to hide from the host immune system. E.g.Borrelia burgdorferi
the spirochete that causes Lyme disease, reveals a region of the genome
that is highly variant among isolates. It has been proposed that a 17 bp
repeat is a recognition site for a ‘cassette’ mechanism by which the
spirochete inserts sequences into its surface protein. These repeats
illustrate that information modulating genetic variation may be contained
not just in the sequences themselves, but in the relationship between
neighboring sequences ( in the case of repeats) and or other
sequence-dependent effects on DNA structure. Cone snail genomes rapidly
evolve diverse toxins. This variation include base changes and
insertions/ deletions, that appears to be intrinsic to the gene sequence,
not due to selection for each individual toxin variant. Olivera’s
observation of a 10 fold higher rate of change between ‘synonymous’ codons
in the exon that encodes the toxin compared to the neighboring exon, and
suggests an "unconventional hypotheses for these unusual data merit
serious consideration".

Constraints may have evolved at the DNA level to protect a patch of
protein sequence that is essential, or of different role or for multiple
roles. Mutation may be ‘silent’ at the level of protein structure, yet
strongly influence the behaviour, and indeed the fate, of DNA.

Degeneracy of the genetic code.
There is more than one way to specify an amino acid, additional
information can evolve within a protein coding sequence of DNA. Shapiro –
" DNA sequences are rich tapestries of information…We have learn to read
DNA more as poetry that as expository prose. Each line in the text can
convey multiply meanings, and they are all biologically important. As our
understanding of genomes becomes more sophisticated, we will increasingly
be able to recognise module boundaries and variation-targeting sequences,
and to predict enzymatic consequences for a specific DNA sequence as
readily as we can recognise sites for cleavage by restriction
endonucleases. Recognising these sites in not straightforward and
requires attention to the relationships between sequences, not just the
sequences themselves.

Genomes are not quiet, stable, repositories of unchanging information,
protected from enzymes that do anything but the most faithful base
duplication. Prescott’s work with the protozoan Oxytrichia reveals
genomes that appear scrambled and ridden with tiny inserts, which relocate
on speciation – demonstrating that DNA sequence can be altered in a way
that is massive non random and yet most importantly, regulated. This
ciliate completely reorganises its genome in the formation of a new
macronucleus in the cell generation after mating, fragmenting chromosomes
into thousands of pieces and then rapidly assembling a subset of its DNA
in an ‘unscrambled’ order. Thus is manages a massive organisational,
indeed, computational, problem. The genome can manage its affairs in ways
that we have hardly begun to fathom. Ripley foresees ‘smart’ sequence
alignments and phylogentic comparisons, which incorporate our knowledge of
molecular processes that drive genome variation and our knowledge of DNA
context effects on the rate and nature of genetic change…to gain this
knowledge, it is important to study naturally occuring nucleic acid
sequences, acted upon by their endogenous enzyme systems.

NEW TOOLS – GM?

We are not the first genetic engineers…many biochemical tools still await
discovery, for we have examined only a small window into natures
creativity. We have had but a glimpse as to what genomes can do and
should be humbled by how much we do not yet understand. Organisms ‘learn’
from each other.. the exchange of genetic information among living
organisms through HGT, demonstrates a potential genetic connection among
all living things and should lead us to treasure and protect nature’s
library of biodiversity.

(000110B7)