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Date:

July 2, 2000

Subject:

antibiotic resistance

 

AgBioView - http://www.agbioworld.org, http://agbioview.listbot.com

Dear Monalisa,

I can provide some of the answers to your query. First of all there are two
kinds of genetic markers used in the production of transgenic plants. The
first are screenable markers. These genes allow one to directly observe the
expression of a transgene. Commonly these are GUS (turn expressing cells
blue), luc (firefly gene which causes the plant cells to glow) and gfp
(jellyfish gene which also causes transformed cells to glow). There have
been a number of attempts to use the latter two genes to select for
transformed cells or sectors and to then selectively subculture these
regions (in tissue culture). This has not proved to be very effective as
the transformed cells are quickly overgrown by untransformed cells (they
are greatly outnumbered by untransformed cells). It seems that success
requires a greater level of selection than can be applied by an
enthusiastic scientist. The second type of marker is what is known as a
selectable marker. These include genes that confer resistance to
antibiotics or herbicides and are commonly isolated from resistant bacteria
or plants. These genes do give transformed cells enough of an advantage to
survive and proliferate whilst untransformed cells are either prevented
from dividing or are killed. Why use antibiotic resistant genes? Because
they work. Not all selectable agents are useful for every plant. In some
antibiotics work well and in others herbicides work well. We choose our
selectable marker based on both availability of the genes (for example it
is just about impossible to get hold of the glyphosate resistant gene used
by Monsanto) and how efficient the selectable agent is at killing cells in
the particular crop one is working on. For example peanut cells are
particularly resistant to kanamycin but are very susceptible to hygromycin
whereas kanamycin is fine for tobacco. There are metabolic/phenotypic
negative selection markers such as the ipt and codA genes but they are not
useful for most plants.

How can we get rid of these genes after selection is complete and we have
our transformed plants growing nicely in the glasshouse? There are a number
of strategies that have been used successfully. Most are based on the fact
that it is possible to cross the transformed plants with untransformed ones
transferring the gene of interest (eg pest resistant gene) but leaving the
selectable marker behind. This is because if genes are present in the
genome at different places they can be separated by traditional breeding
and selection. One approach is to use separate plasmids and directly
transfer them to the plant using microprojectile bombardment
(cotransformation). Because they are separate plasmids they will integrate
randomly at different sites in the plant cell genome. Another is to place
two T-DNAs in a single binary vector with one T-DNA containing the desired
trait and the other the selectable gene. The T-DNA will integrate
separately and randomly as above.

A more high-tech approach uses either the cre/lox, FLP/FRT or R-RS systems
(see Ow and Medberry, 1995). In this case one transforms the plant with a
plasmid containing a sequence (lox) flanking the selectable marker. The
transformed plant is re-transformed with a gene (Cre) that recognises and
excises all the DNA between the lox flanking sequences. The second
selectable marker and its cre gene are then removed using normal meiotic
segregation.

The above methods are limited to plants which reproduce sexually but
transgenes can be removed from vegetatively propagated plants (GST-MAT
system; Sugita et. al., 2000).

These high-tech methods can potentially remove selectable markers from
transgenic plants but may be limited in scope. They require transformation
systems that are very efficient which normally means tobacco and
Arabidopsis. The most practical is probably the two T-DNA system.

However I consider these approaches to be rather academic because I don't
see why there is any concern about antibiotic resistance genes in plants.
As others have written on this site (see Tony Trewavas excellent reply to
Angela Ryan) there is absolutely no reason to fear selectable markers in
transgenic plants. These are naturally occurring genes that most of us have
probably already eaten many times. Antibiotic resistant bacteria are
ubiquitous. Antibiotic resistant bacteria only proliferate and profit in
the presence of antibiotics. Unregulated and indiscriminate use of these
drugs is the problem. This has nothing to do with plant transformation at
all. Eating plants containing these genes will not confer antibiotic
resistance to enteric bacteria (perhaps theoretically possible but at
astronommical odds) and even if it did it would be only one of many
antibiotics effective in treating bacterial infections.

I am sure that eventually plants will be routinely transformed using
selectable markers that are not antibiotic or even herbicide resistance
genes but that time has not quite arrived. In the meantime there is no harm
in using the current crop of markers so why not?

I hope this short explanation is of some use. A few references: Qin et al.,
PNAS 91:1706-1710 (1994); Sugita et al., Plant Cell Reports 18:941-947
(1999); Sugita et al., The Plant Journal 22: 461-469 (2000); Ow and
Medberry In: Critical Reviews in Plant Sciences, CRC Press 14: 239-261
(1995); Odell and Russell in: Homologous Recombination and Gene Silencing
in Plants, 219-270, Kluwer Academic Publishers (1994); Stuurman et. al.,
Plant Molecular Biology 32: 901-913 (1996); Gleave et. al., Plant Molecular
Biology 40: 223-235 (1999); Russell et. al., Molecular and General Genetics
234: 49-59 (1992). There are more but this should get you started.

Malcolm Livingstone

a At 16:50 30/06/00 -0500, you wrote:
>AgBioView - http://www.agbioworld.org, http://agbioview.listbot.com
>
>Dear Professor Prakash,
>
>My name is Monalisa Ghose. I am doing some research regarding selectable
>markers, and would like to use this fabulous listserv/mailserv to present
>some questions about selectable markers to your vast mailserv audience, as
>because I am seeking some answers from the experts, industry representatives
>and so on.
>
>As I mentioned before, my questions pertain specifically to selectable
>markers. Here they are, in no specific order of urgency:
>
> 1. Why are some selectable markers used more often than others?
>For example, antibiotic resistant genes are used as selectable markers
>in the production of numerous crop plants-- why would antibiotic resistance
>genes be used instead of metabolic or herbicide tolerant markers? Why not
>use phenotypic markers?-- leaf colour, shape, etc.
>
> 2. Can selectable marker genes be used in all types of cells--
>animal, plant, human, bacterial, or otherwise?
>
> 3. The technology whether it be selective breeding or recombinant
>DNA, is present to remove selectable marker genes. Why are they not
>removed? (Please don't say because it is not significant to human health,
>unless you can qualify why) How are selectable markers removed? To
>date, I've only found one paper : Gene Transfer with Subsequent Removal
>of the Selection Gene from the Host Genome. Dale, E.C. and Ow, D.W.
>Proceedings of the National Academy of Sciences of the United States of
>America 88, 10558-10562. September 4, 1991
><www.phas.org/cgi/repreint/88/23/10558.pdf> which addresses this question
>directly. Are there any additional papers your subscribers can
>recommend?
>
>These are all the questions I have for now. I look forward to hearing the
>responses to my enquiries. Thank you and all your subscribers for your
>help in advance.
>
>Sincerely,
>Monalisa Ghose.