How to clone an ion channel: to be read in conjunction with the PowerPoint
slides on “Finding the sequence of an ion channel”
Slide 26
We used to (1970s till mid 1980s) sequence proteins by directly identifying the amino
acids starting at the N terminus. But this only worked for very short sequences (up to
about 10 amino acids).
It became easier to get the sequence of DNA or (indirectly) messenger RNA. This
makes sequencing much easier, as shown inthe other slides.
The illustration shows the basic mechanisms of protein synthesis, which will be
familiar to you. Chromosomal DNA sequence determines the sequence of messenger
RNA, which determines the protein sequence.
Slide 27
A reminder of the “genetic code”. The messenger RNA (mRNA) strand is
“complementary” to the DNA. Each group of three nucleotides (called a codon) in the
messenger RNA codes for one amino acid. The first one here (reading from the 5’ end
of the mRNA, placed at the left) is methionine, coded by AUG: this is the first amino
acid in all proteins (unless it is later removed), so that AUG can be considered as the
“start codon”.
In the process of translation, a ribosome “searches for” the first AUG codon (usually
the first, although there are a few exceptions) then attaches amino acids in sequence,
one amino acids for each group of 3 nucleotides.
Slide 28
To work out the mRNA sequence, we make use of the fact that all mRNA molecules
get a “tail” of multiple adenosines (AAAAAAA) added to them. So if we use a short
stretch of TTTTTTT attached to a bead, we can purify mRNA. This step is not always
necessary because the next step also uses the TTTTTTT.
Slide 29
Now we use the mRNA sequence as a “template” to make a strand of DNA
complementary to the mRNA. We use a TTTTTTTT “primer” (a primer is a short
DNA [rarely RNA] molecule that is used to start the process of nucleic acid synthesis)
and the enzyme “reverse transcriptase” (actually it’s RNA-dependent DNA
polymerase, but the name “reverse transcriptase” was coined for it and it stuck).
Reverse transcriptase uses the RNA (brown) to determine the sequence of a new
strand of DNA (blue). Because this DNA strand is complementary to the mRNA, it is
called complementary DNA or cDNA.
The RNA-DNA hybrid in the middle of this slide is used as the basis for a doublestranded DNA molecule: the first strand of the DNA is used as the template to make
the “second strand”, which is complementary to it.
Slide 30
To make a lot of this sequence, it is incorporated into a plasmid, which is put into
bacteria (a plasmid is a little circular piece of DNA which the bacteria can copy and
pass on to their descendants). “Colonies” of bacteria grow from each single
bacterium, and the whole colony expresses the same plasmid. Because the bacteria are
identical, the colony is referred to as a “clone” and the process just described is
known as “cloning”.
Slide 31
The collection of colonies is “screened” to find which colony or colonies is/are
expressing the sequence of interest. That colony can be grown on a large scale and the
plasmid isolated.
Slide 32
The plasmid, once isolated, can be used to make mRNA by a process called “in vitro
transcription” - transcription because we’re using a DNA sequence to make RNA; in
vitro (Latin - “in glass”) because it happens in a plastic tube rather than in a cell.
The mRNA can be injected into oocytes (egg cells) from the toad Xenopus laevis.
These are very large cells (~1 mm diameter) and easy to record. Alternatively (not
shown), and more usually nowadays, we use mammalian cultured cells instead of
Xenopus oocytes, and just inject the plasmid, leaving the cell to make the mRNA.
Injecting the mRNA into oocytes, or transferring the plasmid into mammalian cells,
will cause theoocyte or cell to “express” (i.e. synthesise) the ion channel or other
protein that’s coded for by the mRNA sequence we started with.
Slide 33
The “screening” in slide 6 assumes that we actually know what sequence we are
looking for. This is not an easy step when it is being done for the fisr time with a
novel protein. The example we will look at is the Drosophila potassium channel
called Shaker.
Slide 34
The Shaker mutation of Drosophila causes flies to shake their legs when anaesthetised
with ether. Physiological experiments on these flies indicated that the Shaker mutation
affected an ion channel. Genetic method in Drosophila allow us to identify what
region of a chromosome is affected by a given mutation, and to isolate short
sequences of DNA known to be from these regions. So it was known that the Shaker
mutation affected a region of a chromosome (the X chromosome) near to a known
DNA sequence called 16F.
Slide 35
Starting with a DNA clone from 16F, a “library” (a collection of clones of
chromosomal DNA) was probed to find matching sequences. Once a matching
sequence was found (which would be a stretch of DNA a little further along than
16F), part of this new sequence was used a a probe, which moved the known region a
little further along, and so on.
This is known as a “chromosome walk” because we are “walking” along a
chromosome.
Slide 36
A graphical representation of the “chromosome walk” technique. The result is the
sequence of a long stretch of chromosomal DNA. This is not in itself the DNA
sequence that codes for the ion channel, because it contains both “exons” (sequences
that code for the ion channel) and “introns” (non-coding sequences, that are cut out of
the mRNA before protein synthesis).
This sequence is used as the basis for making “probes (short sequences of DNA) that
are used to search a second library. This second library, instead of being prepared
from chromosomal DNA, is prepared from messenger RNA by the method shown in
slides 3-6. The DNA sequences present in this second library thus correspond exactly
to mRNA sequences that directly code for all the proteins that the fly is expressing.
When we isolate a clone from that library, it is thus a clone containing coding
sequence for the Shaker potassium channel. Several clones would be isolated, and
among the longest we would hope to find a “full length” clone.
Slide 37
Once the “full length” clone is isolated, we get the full cDNA sequence (the “second
strand” sequence from slide 4 is usually the one that is shown - it is equivalent to the
mRNA sequence, except that uracil in RNA appears as thymidine in DNA, so all the
T’s here would be U’s in the mRNA).
Slide 38
We can “translate” the sequence in slide 12, knowing the “genetic code”, to give the
amino acid sequence of the ion channel, as shown in in the lecture.