BOT340, molecular tools In the subsequent paragraphs we will briefly…

BOT340, molecular tools

In the subsequent paragraphs we will briefly summarize molecular biology tools that are
being employed in the course papers. Underlined terms are not further explained but can
be found in any molecular biology or genetics textbook.


Isolating developmental genes

Having a mutant and the corresponding gene product is the ideal starting point for
exploring how the gene acts in an organism. It is important to realize that both are
needed. Having only the mutant or only the gene product is a severe limitation. The
following lists a number of recent molecular tools that enable researchers to switch from
mutant to gene and from gene to mutant. As you will see, the most basic technique
underlying nearly all methods listed below is nucleic acid hybridization: the possibility to
localize a specific nucleic acid sequence through a labeled probe of the complementary
sequence. Under suitable conditions, this label will be selectively bind to and mark a
target sequence.




From mutant to gene:

There are two basic strategies to find a gene based on a mutation. Both aim at obtaining a
hybridization probe very close to the gene of interest, which is defined by a mutation.
Once this has been achieved, further strategies have to be employed to verify the identity
of the gene at this location with the mutation-defined gene.


First, map-based cloning.
If the mutation is caused by a conventional mutagen, it will be necessary to localize the
possibly minute sequence alteration in the genome of the mutant. Fortunately, the
mutation can be genetically mapped relative to DNA sequence polymorphisms (Fig.1).
Given a tight genetic map of molecular markers and a large segregating population, a
mutation can be mapped to a chromosome interval encoding only one or few mRNAs.




1
             m                wt
    gene x           gene x


                 a                 b
mol.mark1            mol.mark1




             a       b
Fig. 1.: The two lines with black dots symbolize a pair of homologous chromosomes. The distance between
a gene of interest (x) and a molecular marker (1) is determined by meiotic mapping.
Briefly, a plant is generated by crossing the mutant allele of gene x "xm" that has been generated in a strain
a to another strain b (carrying a wild type allele "wt" at gene x, "xwt"). Since there is abundant sequence
divergence between the two variant strains, a and b, numerous molecular markers can be visualized. These
reveal sequence divergence at fixed chromosomal positions, for example by generating PCR products of
distinguishable length.
Meiosis in an individual resulting from the above cross (depicted in the figure), will produce gametes that
will either contain one of the parental (xm,1a and xwt,1b) marker combinations or, by crossing-over, new
recombinant marker combinations (xm,1b and xwt,1a). If a molecular marker is very close to gene x, barely
any recombinant meiotic products will be produced. By contrast, if the marker is far away from the gene of
interest, or on another chromosome, parental and recombinant marker combinations will be produced at
equal frequency. Standard calculations for meiotic mapping can be applied. Thus, given a sufficiently large
segregating population, molecular markers extremely closely linked to a mutation-defined gene can be
identified.




Second, gene tagging.
The mutation may be caused by the insertion of a piece of known DNA. When plants are
being transformed with exogenous DNA, the integration of this DNA in the nuclear
genome may affect a gene located at the integration site. For example, it may be
inactivated by disruption and this in turn may cause a mutant phenotype. If a mutation is
due to the insertion of a piece of known DNA, a hybridization probe will detect this
inserting DNA in the genome of the mutant plant. The disrupted gene should be
immediately adjacent to this inserted DNA. Although this is an idealized scenario, a
piece of inserted DNA is the suitable tag of a mutation-defined gene (thus, "gene
tagging"). However, compared to conventional mutagenesis, a lot more work is required
to establish an insertional mutant collection.




2
                     f lanking gene               insert ed DNA                                       rest rict ion   sit e

r e st r ic t io n
sit e
Fig. 2: A piece of inserted DNA has disrupted a gene (indicated by a transcript arrow [lower line]. The
insertion can interfere with many aspects of gene expression and function. In this examples it prevents
translation of a meaningful protein (brown box) from the site of the insertion. Further c-terminal proteins
(hatched box) will therefore not be translated. This may render the protein partially or totally non-
functional. Gene and inserted DNA may reside on a single restriction fragment.




Once a hybridization probe adjacent to the gene of interest has been identified by either
the mapping or the tagging method, the closely linked transcription unit may be identified
by inverse PCR or by generating a genomic library of the mutant.
Final proof that the right transcription unit has been identified can be provided by
identifying the mutation in the actual sequence of the mutant, or by normalizing the
mutant phenotype upon integration of a wild type copy of the suspected gene into the
mutant ("transgenic rescue").




From gene to mutant:

It is not part of any of our papers, but it is important to know that the inverse strategy is
also feasible. Targeted mutagenesis (direct replacement of a wild type copy by a mutant
copy of a gene) does not work efficiently in higher plants. Therefore, mutants to a given
gene sequence are identified by "reverse genetic" screening of large populations of
insertional mutants which are expected to contain insertions in virtually all genes. Since
in this case the sequence of the gene of interest is known, primers to its sequence can be
designed. Briefly, a PCR reaction between a primer from the gene of interest and another
one will only result in a PCR product, if the two primers are located adjacent to each
other (~1kbp; see Fig.3). Therefore a successful PCR reaction indicates the integration of
the insertion DNA into a gene of interest. Because the PCR reaction is extremely
sensitive, the DNA of hundreds of insertional mutant lines can be pooled. Insertion in the
gene of interest will remain visible as a PCR product. This line can subsequently be
identified within a positive pool.




         f lanking gene               insert ed DNA




3
Fig.3: reverse genetic screening of insertional mutations. Binding of a primer to the known sequence of the gene of
interest (top row, left) and of another primer to the inserting DNA (right) will only produce a product, if the insertion
has occurred in the gene of interest.




Visualization of gene expression profiles



Visualization of reporter gene expression:




 r eg ul.




 r eg ul.




                       report er gene


If it is possible to identify the regulatory sequences that are sufficient to confer a gene's
mRNA expression profile, one can replace the actual coding sequence partially or totally
by the coding sequence of a reporter gene. A reporter gene is a gene that can more
conveniently be visualized. It may be fluorescent or may catalyze an enzymatic reaction
towards a visible compound. Note that a reporter gene expression profile may not
accurately reflect the gene expression domain, if the regulatory sequences used in the
reporter gene construct were insufficient or if the reporter gene product and genuine gene
product have vastly different stabilities.




Visualization of mRNA expression:




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Distribution of the genuine transcript of a gene of interest in a tissue section can be
visualized by hybridizing a labeled probe to the fixed sectioned material in a small
amount of liquid on the microscope slide. Later this hybridization solution is removed
and the position of the hybridized label is visualized by methods that depend on the type
of label.



                lab e lled
                probe
                                            f ixed   t issue

                 s p e c if i c                                 microscope       slide
                 mRNA


Visualization of protein expression:

To visualize the distribution of the protein product of an identified gene, the gene product
must first be produced in sufficient amounts to generate a specific antibody against this
gene product. If this antibody binds to its specific protein target in a tissue sample (not
necessarily a section), a second (commercially available) antibody recognizes epitopes in
the constant region of the first antibody. This second antibody carries some sort of label
that can be visualized.



            lab e lled
            2 nd ab



                   1 st ant ibody           f ixed   t issue

                 s p e c if i c                                 microscope       slide
                 m RNA
                  protein




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