The DNA Series:
Hello everyone, this is going to be a series of blog posts. Stay tuned so you don’t lose a single one! For this series, the blog posts will be:
- Understanding the code of life
- Conventional breeding
- Mutagenic breeding – a) Introduction to mutation
- Mutagenic breeding – b) Induced mutation
- Transgenic breeding
- Interference RNA
- Gene editing
Mutagenic Breeding: Introduction to Mutation
The word mutation usually carries a strong feeling, but in reality a mutation is a change in the DNA sequence of an organism. We have discussed on the previous blog that living organisms have DNA, and that DNA carries the code to an organisms’ characteristics. What you might not know yet is that nature can make mistakes sometimes, and those mistakes are called mutations!
Every new biotechnology comes from an observed behavior of a living organism, and for mutations, that is also the case. To make it more understandable for favorable crop breeding, we must rewind a bit and acknowledge how and why mutations occur naturally.
DNA Replication and Repair
When the cell of any living thing is preparing for division, DNA replication starts with the help of many enzymes. First, the DNA double helix is unwound and separated by topoisomerase and helicase, respectively. Each strand then serves as a template for DNA polymerase to synthetize a new, complementary strand (i.e. in the DNA, adenine (A) always pairs with thymine (T), and guanine (G) always pairs with cytosine (C)). Note that instead of north, south, east, and west, the DNA has its direction recognized by a 5’ → 3’ system; the enzyme DNA polymerase can only synthesize DNA from 5’ to 3’ direction.
When DNA is replicated, errors can occur, but they are extremely rare. The initial error rate of DNA polymerase is about 1 mistake per 100,000 nucleotides synthesized. However, cells are equipped with proofreading and mismatch-repair systems that correct almost all of these errors. After these correction mechanisms act, the real error rate drops to about 1 in 10,000,000,000 (ten billion) nucleotides. To put this into perspective, let’s consider Arabidopsis thaliana, a common model plant. Its genome contains approximately 2.7 million nucleotides per cell. With the corrected mutation rate, you would expect one mutation to occur for every:
In other words, a single mutation in Arabidopsis typically appears only after thousands of replication cycles.
The first time that the replication is checked for errors is by DNA polymerase itself, which discriminates against the incorporation of mismatched bases. Proofreading, also made by DNA polymerase, is another important mechanism for correction of errors in DNA replication. After finishing the replication of each strand, DNA polymerase goes through the complementary strand again to check for any errors. Even with all that scheme to repair mistakes if they happen, there is still a chance that an error goes through.
Now we understand that mistakes, or so-called mutations, can happen naturally. But how do they affect an organism? ↓
Protein Expression
Proteins are complex molecules that are constituted of amino acids, and amino acids are constituted of a codon, which is formed by three base pairs (here, uracil (U) takes thymine’s (T) place). They can have roles in the structure, function, and regulation of the cells of a living organism. In plants, for example, proteins have roles in photosynthesis, growth and nutrition to seedlings and plantlets, structural support, cell transport, ion regulation, etc. Since every three base pair forms a codon and signals an amino acid and amino acids come together to form a protein, the recipe of a protein is given by the DNA sequence.
To form a protein, the DNA sequence must go through transcription, where the DNA is copied into a RNA molecule, that generates a messenger RNA (mRNA) to carry the genetic sequence. In passing from DNA to RNA, one nucleotide changes: where would be a Thymine (T) in DNA will be an Uracil (U) in the RNA. The mRNA is then translated into a protein.
Types of Mutations
With that in mind, if there is a change in the DNA sequence, there will be a change in the mRNA and the resulting protein. Mutations can occur in different ways, depending on the change that the DNA has suffered. The mutations can be classified into point and frameshift mutations.
In point mutations, one nucleotide is changed. This one nucleotide change in the DNA can be silent, meaning that it would not affect the resulting protein (this is possible because the same amino acid can be formed with different nucleotides at the end of the codon); it can be nonsense, in which case the change will create a stop codon, so the transcription will not continue, leading to the protein not being formed at all; and (conservative and nonconservative) missense mutations, in which the nucleotide change is in the middle of the codon. In the conservative missense mutation, the change in that one nucleotide will result in a change in the amino acid, but this new amino acid will have a similar molecular structure as the original one, keeping the protein functional. The nonconservative missense mutation will result in a change of amino acid that is completely different from the original, leading to a non-functional protein.
Another type of mutation is frameshift mutation, which changes nucleotide(s) in the sequence by deleting or adding it. This causes a shift in the reading frame. As an example of an addition, let’s say an Uracil is added after the start codon “AUG”. Instead of the next reading codon be “AAG” which is a Lysine, the next reading codon will now be “UAA” which is a stop codon that signals to the enzyme that the translation should stop there. In this case, no protein will be formed, causing a serious effect. Another example is if we have a missing Uracil after the Lysine codon (AAG). Here, instead of the third codon being “UUU” which is Phenylalanine, it will be a Leucine (UUG). This could result in changes in the protein if the amino acids do not have similar structures. Now, if a whole codon is missing, there will be one less amino acid in the protein, causing it to malfunction.
Natural Mutations
Naturally occurring mutations have been very important for the evolution of all species for 3.5 billion years. Mutations can be deleterious, meaning that the resulting protein of the mutated gene won’t be produced, or it is produced but not functional, or even produced without normal functionality. Mutations can also be neutral, in which case it won’t have significant effects on the plant’s fitness, survival, or reproduction. Mutations are not aways bad as one may think. Beneficial mutations are those that make the organism more adapted to its environment in comparison to its parents.
As an example of a deleterious mutation, albinism mutation in plants usually means no chlorophyll (the pigment responsible for photosynthesis) is produced. Without photosynthesis, the plant won’t be able to produce energy and, therefore, die. Examples of beneficial mutations in plants include the removal of a toxic compound amygdalin in sweet almond kernels, caused by a point missense mutation; the longer grain length in rice, caused by a point nonsense mutation; and the six-rowed spikes in barley, caused by a frameshift mutation.
Natural mutations are primarily triggered by errors during DNA replication and repair, and exposure to natural environmental factors. The mutation rate is known to be affected by radiation, temperature, moisture, age of the seed, viral infection, and chemicals.
Now that we understand how natural mutation occurs, it is time for the next step: mutational breeding! The next blog of this series will dive into the world of induced mutations. How are we using this natural occurrence in our favor when developing new crop varieties? See you next month.
