Effect of chemical mutagens

Effect of chemical mutagens

Chemical mutagens affect the organisms at molecular level. Action of chemical mutagens may or may not give beneficial result. It can change the sequence of nucleotides or may replace the nitrogenous bases in DNA. On the basis of effect of chemical mutagens (at molecular level), it is classified into following categories. We have to understand the molecular basis of to know its effect.

 Molecular basis of mutation: - Mutation at molecular level changes the normal nucleotide sequence.              

Replacement of a purine(A or G) with the other purine or a pyramidine(T or C) other pyramidine is known as transition where as replacement of a purine base with a pyramidine base and vice versa is said to be transversion. These two mutations show a phenomenon of substitution and occur at DNA level. Another type of mutation is frame shifting which includes deletion and addition. The nucleotides are arranged on DNA normally as 

Mutation may occurs at larger or smaller level hence on this basis, it is classified into following two classes i.e.

 Macroalterations are large changes, such as duplications, deletions, inversions or rearrangements of a large number of bases.
Microalterations involve single base pairs.

 Another classification of mutations has to due with multicellular organisms where cells might belong to the germ-line, if they contribute genetic information to future generations; or, if part of the body that will never contribute genetic information to future generations, the somatic cell line.

• Somatic mutation is one that will never contribute to the germ-line of the effected individual.
• Germinal mutation is one that occurs in a cell that is a progenitor to a germ cell(s).
• For example, if a human embryo sustains a mutation in a cell that later becomes a germ cell: a sperm or egg, it is a germinal mutation.
• Otherwise, it is a somatic cell mutation.

 Another classification system for mutations has to do with using the "normal" or most common state as a reference point. The normal state is called "wild-type".

                                                                                                                                                                       *      The change from wild-type to mutant form is called a forward mutation.

• The change back to wild-type is called a reversion or back mutation.
• A reversion can occur either by true reversion, which is when the DNA is restored to the exact previous form;
• or, by a suppressor mutation, when the wild-type phenotype is restored, protein function is restored, but the DNA is not back in its original form. Can you think of a way that a suppressor mutation might occur?
• Reversions are random events. A forward mutation is caused by changing anyone of the bases, or by adding or deleting bases. However, a true reversion is when that exact change is reversed. Therefor it makes sense that the relative frequencies for these classifications of mutations are as follows:
  Forward mutations > suppressor > true reversions.

Another classification of mutations is based on the functional effects of the change.

·         Morphological Mutants have altered shape.

·         Lethal Mutants die as a result of having the mutation, can be dominant or recessive, but most often are recessive. This makes sense because there are so many enzymes an organism must have to live; if both copies are defective, the organism would fail to survive.

·         Conditional Mutants are normal under one condition (permissive), but abnormal under another (restrictive). These are extremely useful for studying processes such as development and DNA replication.

·         Biochemical Mutants cause defects in biochemical pathways for a substance, which is then deficient.

·         Loss-of-Function mutations cause a loss of function that is found normally in wild-type. 

·         Gain-of-function mutations create a new function not normally found in the wild-type. Hairy-faced people would be an example

DETECTION SYSTEMS

How can we observe mutations when they occur?

• Haploid organisms are extremely useful for studying mutations and mutagens. When mutations occur, they are often observable. What mutation any haploid organism gets, it expresses, because generally there is only one copy of each gene.
• Diploid organisms are tougher. Recessives, the most common type of mutation, are often invisible. They are masked by the presence of a dominant allele.
• One approach is to use F₁s. The sudden appearance among the F₁ of the recessive phenotype provides a measure of mutation rates.

• In Paramecia, autogamy can be used. Extra credit will be given if you can accurately recount the class lecture coverage of autogomy in Paramecium tetraurelia and its usefulness for mutagenesis studies.
• In male humans and Drosophila, genes on the X can be observed. So, the mutation frequency can be determined by studying males.
• There is a well developed system in Drosophila that allows a certain X to be studied. That X is mutagenized in the males, then passed through an F₁ female and then to the next generation of males.

·         The Genetic Basis for Bacterial Antibiotic Tolerance

·         Replica Plating is a method for screening bacteria after selection. It involves transferring bacteria from discreet colonies on a plate to another plate while maintaining their relative positions to each other. The plate transferred to usually contains another medium useful for selection or screening.

·         Before 1952, it was thought that bacteria adapted to living in the presence of substances like antibiotics by developing a biochemical tolerance. In fact, some scientists actually failed to come to grips with bacteria being like eukaryotes: organisms that depend on the use of hereditary information.

·          

·         The Lederbergs' experiment verified that antibiotic resistance in bacteria was do to the presence of genetic variants in the population of bacteria prior to exposure to the specific antibiotic. They also demonstrated that the resistance was heretically transmitted.

·         Replica Plating is still a widely use and useful technique.

Screening for Mutagens: The Ames Assay

So how can we test a new or suspect substance to determine if it is a mutagen?

• There are many defined strains of bacteria available, which each have a known mutation.
• The mutation in one strain might be caused by a certain base substitution.
• The mutation in another might be caused by a frameshift.
• However, in order for a strain to be useful for testing a compound, the kind of mutation responsible for the loss-of-function must be known.
• We discussed before how the forward mutation rate is so much higher than the rate for reversion.

This is not a problem when working with bacteria, because in a short time we can easily grow billions of cells.

• Among the billions will be a few that have reverted.
• If you put a mutagen into the medium your growing the bacteria and the mutagen causes the kind of mutation that is needed to get reversion to occur, then more revertants will be found in that container.
• You put some bacteria into a flask with medium. In the exact same way you place other bacteria into the same medium but include a suspected mutagen. After growing the bacteria in the two flasks for 10 or more generations but to the same populations sizes, you plate out the bacteria onto plates missing the substance that the mutant strain needs. Only mutants will grow on these plates.
• If you find more on the plates that contained the suspected substance, then you have verified it is a mutagen.
• If the numbers are equal, it means that the substance did not cause the kind of mutation needed for that particular type of mutant to revert.
• You must screen a suspect substance with different types of mutations. Screen with bacteria that revert when frameshifts occur to determine if the mutagen causes frameshifts.
• Screen with a base pair substitution of A:T to G:C to determine if this is the kind of mutation the suspect agent might cause.

If treating any of these kinds of strains yields an increase in the number of revertants, you will know what kind of mutation the agent causes.

It was discovered that some substances that cause cancer in humans are not mutagenic to bacteria. This was a puzzle. Ames discovered that if liver microsomes (fragments of smooth ER) were added to the suspected compounds, and then the combination was added to bacterial strains, the reversion rate increased.
What does this mean?

• It means that the compounds themselves were not mutagens, but the liver changed the compounds into mutagens, in the process of breaking them down.
• The Ames Assay is an assay like what is described above, but often including liver microsomes, to test whether the breakdown byproducts are in fact mutagenic.

Cancer: Mutations can cause cancer. Proto-oncogenes are genes that help regulate cell proliferation. Mutations can make things go a little crazy. Cells dividing out of control leads to tumors.
Mutations can be useful to mankind's evolution as well. But mutation induction for generation of useful mutations works best for organisms with high reproductive rates, and certainly not for humans.