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[SANET-MG] replies to more questions by Douglas on high ethanol yeast
Enclosed below are some more Q&A on high ethanol yeast
Douglas Hinds wrote:
I have tried to explain about yeast genetic manpulation in reply
Thank you, Professor. However, your explanation have raised a few more
*Legitimate Recombination and Site Specific Mutagenesis*
* Site specific mutagenesis: *The process involves changing specific
DNA code words in a particular gene. In the case of the high ethanol
yeast a commercial kit called the quick change site specific
mutagenesis kit was used. Unfortunately the kit is not yet available
ans:the kit is described at
Its use was listed in the methods section of the paper on gm yeast.
Commercial products are used a lot in genetic engineering Off the shelf
technology is commonplace.
Please confirm that "a commercial kit called the QUICK CHANGE SITE
SPECIFIC MUTAGENESIS KIT" was actually used.
ans: The DNA code has four letters and the organisms entire code is
known so that altering the specific amino code words just means changing
a the letters A,T, C or G , a mutation may result by a change from A to
G or T to A etc. thus a specific amino acid change in a protein can be
engineered. Cranking out oligonucleotides is fairly commonplace and
usually it is fastest to but the altered sequences from a supplier than
to depend on a graduate student just learning his or her job.
If so, does it's being a commercial kit mean that the mechanisms the
kit employs are secret, or can they be explained? The methods are nver
secret and well explained on the box the kit comes in. Sectecy was not
allowed in science publications until corporations bagan to control
publications. However, patents require full disclosure and our a good
source of honest information even though many science journals are no
longer very truthful as a policy (consider the British Food Journal).
If they have been patented, more details should be available. ans:Yes
that is the case and the patent application is also truthful because
it must be.
Short DNA chains (oligonucleotides) with desired mutations were
first prepared by the experimenters or purchased at the
Whether the desired oligonucleotides were prepared or purchased
(IOW, prepared by someone else), what specific process is used to
create the desired mutations? (Perhaps you explain that further on).
The quick change kit provides the tools for changing the wild type
gene born on a bacterial plasmid.
You are saying that the gene insertion tools provided by the Quick
Change Kit involve the use of an invasive bacterial plasmid? Just
the plasmid? Not the bacteria itself? If not, how does the plasmid
enter the cell?ans: Just the plasmid, you cannot do the job while the
bacterium is wiggling about. Transformation means that once the
plasmid is modified tou need to mix it up with bacteria in a kind of
cocktail of chemical which encourage the bacteria to take up the plasmid.
The mutant oligonucleotide is annealed to the wild type plasmid
How does the annealing occur, and how are the plasmids removed from
the wild bacteria?
ans: to remove the plasmid you need to dissolve(kill) the bacteria snd
purify the plasmid using a technique called electrophoresis or a
centrigugation technique.To anneal the oligonucleotide to the plasmid
you need to melt the DNA to separate the strands then anneal it at a
controlled temperature. Then the annealed DNA is replicated using the
special DNA polymerase in the kit.
ans; Plasmids are not organelles they are just plasmids.;I agree with
Lynn on anything she wants. Lynn and I were graduate students in the
same lab at Wisconsin (she was at that time married to Carl Sagan) I saw
Lynn for the first time in 46 years last spring. She was as sweet as
Lynn can be.
Since plasmids are organelles, they are self contained entities with
their own DNA which reside inside the cells of complex organisms
(eukaryotes). Is that correct? Do you agree with the conclusions of
Lynn Margulis regarding the origins of eukaryotes?
ans:Homologous (legitimate) recombination just means the run of DNA
undergoing recombination must be homologous (identical) with the run of
DNA with which it recombines. What else is down or upstream of the
recombination does not matter.Bacteria like other living organisms
harbor diversity but all that is need for homologous recombination is
and the quickchange DNA amplifies the annealed plasmid to produce
plasmids with specifc mutations specifying altered amino acids in
the protein regulating the ethanol network in yeast. Next, the
mutant gene must be inserted into the yeast at a specific locus.
That step invovles legitimate recombination.
*Legitimate Recombination:* Genetic engineering bacteria and yeast
(and other fungi) is achieved using legitimate (homologous)
Does homologous recombination indicate that no foreign
(inter-species) genes are involved? IOW, does homologous
recombination involve only intra-species genes? If so, does the
integrity of the individual organism's own genome have any meaning
to geneticists in this context? Are all bacteria of a given variety
genetically identical 100%? IAC case, as I recall, genes from yeast
varieties in other countries were inserted.
ans: In genetics congeners are organisms with very similar genomes,
except for a small fraction. For example, recombinant congenial mice
strains are produced in laboratories as a tool to study disease.The
inserted genes are not congeners and not necessarily homologous except
for the inserted tails. The inserted gene disrupt the function of the
target gene. The process is also called allele replacement but that term
is normally applied to insertion of functional alleles rather than a
mutant allele of another locus.
Genes to be inserted require require short tails from the gene of the
insertion site to target the gene insertion into a chromosomal locus.
How are those short tails copied/attached to the genes to be
inserted?ans: the DNA tails for homoogy are added to the DNA sequence
by a process called ligation, ligation kits have been available for
forty years or so.
The inserted gene disrupts the target gene allowing rapid
selection [of] the inserted gene.
So the inserted gene is a congener of and competes with the target
gene. That is the way most antibiotics work.
In the case of the yeast ethanol control gene targeted to the
uracil locus the transformed yeast colonies would be identified
using replica plating on uracil lacking media. The colonies
bearing the disrupted gene do not grow on the uracil minus media
but leave a ghost like pattern on the agar.
So yeast bearing the original gene require uracil and the yeasts
that assimilated the genetically engineered gene in the correct
location do not. (Please confirm). IOW, the creators of the mutant
yeast are using a method intended to insert the modified gene in a
specific location but are NOT able to carry out an insertion process
that guarantees that this actually occurs, at present. All they can
do is verify after the fact that it did or did not occur. This
suggests to me that "secondary" modifications may be occurring
concomitantly, and that only the "goals" are being looked for. Since
any "secondary" modifications may be occurring concomitantly are
irrelevant to the creators' goals, these are NOT being looked for.
The colonies modified with the regulator gene are picked off the
uracil master plates and grown up for fuller testing.
ans: the technology is fully understood but the impact of inserting the
mutant gene needs fuller study.
This too suggests that the full effects of the process that was
employed -that which actually occurred- are not yet fully
ans: legitimate and illegitimate recombination are any thing but
anthropomorphic , the terms have been used to describe the different
modes of recombination in microbes for over fifty years. Legitimate
recombination depends on DNA homology while illegitimate recombination
ignores homology, as in crop genetic engineering, genes are inserted
during random breaks in the chromosomes of the crops. Legitimate
recombination would greatly improve crop genetic engineering but good
systems have not been found except for one non-crop plant. I suggested
that the products of illegitimate recombination should be designated to
be bastards, the German plant breeders always called hybrids bastards in
the scientific papers. I have not suggested those producing GM crops
should be called bastards, a simple sob should suffice :-) .
Crop biotechnology does not use legitimate recombination but
instead requires illegitimate recombination in which the gene to
be inserted slides into a double strand break in the DNA.
The crop genetic modifications truly produce bastards which are
the products of illegitimate recombination.
"Legitimate", "illegitimate" and "bastards" seem like rather
anthropomorphic concepts in this context, which carry a perhaps not
fully deserved load of connotation. IOW, just how legitimate is
legitimate? Is it simply closer to being legitimate than
ans:ans: I disagree that redundancy does not normally take place in
evolution. In both plants and animals gene duplications at separate loci
lead to chromosome rearrangements following recombination. The
consequences are different depending on whether the genes are on the
same or different chromosomes. Such duplications are usually
debilitating but do lead to rapid diversity and may contribute to
speciation. These impacts have been studied for nearly 100 years.
Animal genetic engineering employs both legitimate recombination
and illegitimate recombination. Illegitimate recombination tends
to make the chromosomes of the recombinant crop unstable in part
because of the needed array of control and selection genes needed
to run the desired gene insert.
While any additional complication would logically augment the risks
and increase the possibility of procuring unexpected results, viral
insertion y gene gun bombardment produce gross results, practically
assuring an imprecise insertion process.
Legitimate recombination is normally stable but when genes
homologous to the inserted gene are already present at another
locus then mitotic or meiotic recombination between the two will
lead to translocations, duplications or deletions.
Redundancy and conflict. This does not occur in conventional
breeding -where evolutionary change is generated from within- rather
than via manipulative, imposed and invasive action.
ans: biotechnology seems to be driven by the rule, what you don't' know
will not hurt you! the only place funding such studies seems to be EU
and US journals seem to not publish bad news for fear of unpleasant
things coming form corporations.
The main concern about the manipulation of network regulators is that
the genes have highly pleiotropic effects. Consequently they may lead
to production of unpredicted toxins.
When multiple functions stemming from a single gene produce highly
variable results in number of tissues, any genetic change is likely
to effect each manifestation of the gene in variable and
unpredictable ways, and a desirable trait may very well wind up
accompanied by an undesirable one.
Such side effects must be looked for.
Is anyone in fact doing that?
ans: I have not said that, I said that the DNA had been sequenced, I had
not intended to imply that PhD are bred from yeast even though that
might be a thought?
On the up side bakers yeast was never a pathogen to anything ,
unless, of course, it was modified with a gene for a toxin.
Modifying the gene in any way could produce an unexpected toxin.
The genome of baker’s yeast is fully sequenced and its genetic
behavior has yielded a grand crop of PhDs and even Nobel prizes
rivaling that of bread mold.
You are saying that not only has the genetic code of baker's yeast
been fully mapped, each and every function of each and every gene
has been determined, as well as the conditions that turn each
function on and off?
If so, the same can not be said regarding the GM crops already on
I see a rush to cash in on technologies and processes that are still
poorly understood, when what is needed -and should be happening but
isn't- is a lot more study, realized from a public interest
perspective. I see a lot of irresponsible activity occurring with
little or no oversight. I see goals and results that don't mesh, and
what I DON'T see is who is going to foot the bill for damages that
are already occurring, without being tabulated. While ignoring their
existence may seem like a solution to some, how long can that last,
and how extensive must the damage become before folks get wise - just
like what happened with tobacco, DDT and finally, the tragic,
grossly bungled and extraordinarily foolish war in Iraq.
I hope that this discussion has helped to clarify the matters and
serve as an introduction to the looming threat of network regulators.**
I believe we are making progress, but important questions remain.
IAC,I appreciate your taking the trouble to discuss the issues in
greater depth with me.
Douglas Hinds wrote:
Hi Joe and other sanet subscribers following this important thread:
Prof. Cummins wrote:
Thank you for the interesting questions. I read the paper in
science along with its accompanying on-line 32 page description of
the genetic methodology used to create the high ethanol yeast.
Before answering your questions I will go into the methodology a
bit. I will mention that I taught Microbial Genetics for over
twenty five years and published in both yeast and fungal genetics
and molecular biology.
The system developed for making high ethanol yeast is certainly
genetic engineering but it is not genetic modification with
Key regulators for production of high level ethanol in the
presence of high level glucose were identified in yeast. Those
genes DNA sequences were altered by a process called site specific
mutagenisis then reinserted into the yeast at a particular locus
(the uracil gene).
Could you describe the process of "site specific mutagenisis" used
to alter the DNA sequences of the high level ethanol production
genes; and explain how researchers were able to reinsert these DNA
sequences into the yeast genome at a particular locus (the uracil
gene) with such great precision?
The fact that they were able to do this seems strange to me, because
as I understand it, when the GE organisms sold by Chem/Gene/Seed
companies like Monsanto and DuPont (which apparently participated in
the creation of the GE yeast referred to), were created, there was
no way to determine where within the genome the inserted gene would
end up, thus shifting the rest of the genome's components in the
process and as a result, making the genome itself unstable. Is that
correct, Professor? And if so, how is the process used by the MIT
group different? How were they able to take out specific genes and
reinsert them into the exact same place?
One significant difference may be the fact that no new genes are
inserted in this process, I suppose. Another significant difference
may be the fact that yeast is a much simpler organism (which
therefore, has a much simpler genome) than the genomes of the plants
cultivated by man. Likewise, more is known about yeast genetics than
is the case with corn, soy, cotton, rice and potatoes etc. Is that
The regulatory genes controlled a number of genes in a metabolic
network for ethanol production and most importantly, [the]
ability to tolerate high levels of ethanol and glucose.
The current work on such key regulators follows work on E coli
ethanol production by modifying a sigma factor regulator of
transcription. Douglas asked "However, we could call the effect I
refer to "genome shifting", which adds an unknown, unforeseeable
and uncontrollable element to the expected results. Do you agree?"
I do not entirely agree.
Yeast genetics are far more precise than the crude methods used in
crop plants and the genes in the yeast have been precisely altered
by mutations on knowingly altered DNA. Manipulating a metabolic
network is new and breathe taking but could lead to unexpected
toxins being produced.
These will have to be checked on in the near future but since the
yeast is not for very strong beer but for commercial ethanol fuel
the toxins could be ignored. A former student and colleague, Inge
Russel, developed a very high ethanol yeast by breeding and
selection alone and her strain is presently better than the
promoted higher tech system because it was bred from a commercial
ethanol yeast rather than into the laboratory strains used in the
high tech version. Lab yeast tend not to do all that well in
commercial operations for brewing, wine making or ethanol making.
The gene regulators are somewhat similar to the MADS box
regulators growing popular in crop genetic manipulation that I
discussed previously. However, the MADS box alterations have been
transgenic while the network ethanol yeast are not.
Douglas asks "To what extent is the GE yeast likely to cross with
native yeasts existing in nature? Is the threat similar to that
created by GE crops, or are GE yeasts likely to be less
threatening? You ask an important question Douglas. My feeling
this that regulators will have a hard time getting their teeth
into genes that are precise;y altered within a particular
If unexpected toxins are produced because of metabolic network
alterations then we are in real trouble. Bakers yeast is not a
pathogen to anything , I believe, but it has been used as a
probiotic. Killer beer is every boy's nightmare! However, the
toxins should show up early and be dealt with promptly or even
ignored for making fuel. However, toxic ethanol yeast in the gut
would not be any fun at all.
Manipulating metabolic networks is brand new but clearly here to
stay. Organic farmers and the industry will soon be faced with
difficult decisions about organic foods. Can genes and networks
manipulated by engineered DNA code word changes within a crop be
considered organic? We will soon have to decide.Let us hope the
decision is ours and not the bureaucrats.
Finally, many recollect that I objected to GM wine and I still do.
That stuff is transgenic and should be labeled, dammit all. You
cannot imagine the abuse I get from the developers of GM wine!
Thanks so much for the important questions, excuse my rambling
Your answers were clear and to the point, Professor. And we are very
fortunate to be able to be able to draw on an information source
coming from the highest possible level, both in terms of your
technical competence and scientific credibility but also (and
very significantly), in relation to the critical social and
environmental factors that come into play in the context of this
And for that all of us here thank you, Joe.
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