In this example alleles differ by the number of tandem repeats of the motif CGAA
Electrophoresis is the single most powerful tool and can be used for DNA and protein.
Electrophoretic mobility of a molecule is the velocity attained in an electric field of 1volt/cm
mobility µ = q/f
Where q is the charge on the molecule and f is the frictional resistence which depends on the radius of the protein (and viscosity of the medium). This relationship means that molecules can be separated on the basis of their charge or because of size differences.
Crude protein extracts can be used together with specific detection systems.
Allelic forms of proteins can be separated on the basis of the differences in surface charge due to substitutions of amino-acids which have charge R groups using gel electrophoresis. Subtle changes in surface charge can be detected by an adaptation electrophoresis known as isoelectric focussing.
Amino acid substitutions in non critical parts of proteins appear to be quite frequent. The first indication of this came from electrophoretic studies of soluble enzymes and serum proteins in the 60’s and 70’s. These proteins were studied first because they could readily be separated by electrophoresis and because they were easy to detect.
While serum proteins were detected by using general protein stains enzymes could be detected making use of their catalytic function.Total cellular extracts are subjected to electrophoresis and specific ‘in situ’ assays are done by placing a reaction mix on the surface of the gel. The idea is to detect a coloured, fluorescent or radioactive product of the reaction. The mix includes the enzyme substrate and various other reagents, such as adjacent enzymes in the metabolic pathway, which will lead to the formation of a detectable product. Several other functional detection systems are used. These often involve binding of specific ligands. Specific detection can also be achieved by making use of antibodies raised against the protein.
Occasionally mutations lead to variant proteins with altered size. These size differences may be detected by standard gel electrophoresis and they may also be detected by an electrophoresis technique which involves denaturing the proteins and binding the negatively charged detergent sodium dodecyl sulphate. This technique separates proteins according to their size because the charge/mass ratio is constant and large molecules migrate more slowly than small ones because of the seiving effect of the gel (frictional resistance).
In this example alleles differ by the number of tandem repeats of the motif CGAA
In all these examples you will note that I have described the phenotype as 1, 2-1 and 2. The phenotype is what you observe, whether it is a pattern of bands on a gel or eye colour. The genotype can be deduced by following segregation in a family. It is always important to consider the possibility of a silent allele (see the ABO system in the previous lecture).
If two or more alleles can be distinguished in phenotype in the presence of the other they are said to be codominant. An example is seen in the ABO blood group where the A and B alleles are codominant.
Silent alleles are ones which fail to code for a functional product and they are usually functionally recessive. In a multiple allele system, it is sometimes not obvious that a silent allele exists. This can give confusing results. Consider for example
A/A x A/B (phenotype A crossed to phenotype AB)
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V
A/A : A/B
1 : 1
and compare with
A/O x A/B (phenotype A crossed to phenotype AB)
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V
A/A : A/O : A/B : B/O
1 : 1 : 1 : 1
It would be important not to lump together these two different sorts of crosses but when there are only small numbers of offspring (which is the case in most human matings) some offspring classes may not be represented in a family and it may not be obvious which type of mating you are examining. Note that both A and B are dominant to O.
Alleles which lead to enzyme deficiency in the inborn errors of metabolism are silent alleles. Silent alleles are usually rare, although this is of course not the case for the O allele of the ABO blood group system.
This occurs where the action of one gene masks the effects of another making it impossible to tell the genotype of the second gene. The cause might be that both genes produce enzymes which act in the same biochemical pathway.
If the product of gene 1 is not present because the individual is homozygous for a mutation then it will not be possible to deduce what the genotype is of gene 2 by looking at the phenotype. The Bombay phenotype in humans is caused by an absence of the H antigen so that the ABO phenotype will be O no matter what the ABO genotype.
Sometimes several similar (or perhaps not very similar) proteins are encoded by separate genes and can fulfil essentially the same functional role - perhaps in different tissues. The genes may have arisen from a gene duplication event or they may share common function by convergent evolution.
This is sometimes because they have a complex structure involving interaction of several quite different polypeptides encoded by different genes. An example of this is the enzyme phosphorylase kinase (which can be affected in glycogen storage disease) and contains subunits encoded by eight different genes some of which fall into two classes. Two encode alpha subunits and are X linked - and thus a defect in these genes leads to an X linked disorder. The others, which encode two gamma, three delta and a beta subunit, are autosomal.
In many cases two or more identical polypeptide chains are associated to form the active, functional protein. These are often called multimers. Proteins made of two subunits are called dimers, of three are trimers and of four are tetramers, etc. Proteins with identical subunits are called homo dimers. Proteins with non-identical but related subunits are often called heterodimers. Phosphorylase kinase is a heterooctomer with a structure 
. Haemoglobin is made up of four subunits, 2 alpha and 2 beta and is thus a heterotetramer..
| An example is lactate dehydrogenase which is made of tetramers comprised of two different kinds of polypeptides. This gives rise to five different isozymes: |  |
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Isoforms can usually be separated by electrophoresis or some other separation technique.
They exist because of :
The existence of protein/protein interactions has functional consequences in relation to disease causing mutations. This can have an impact on the mode of inheritance. This will be covered in further detail in a future lecture.
Examples:
This figure shows the multiple isozymes of phosphoglucomutase in red cells. The patterns observed result from the expression of two phosphoglucomutase genes, allelic variation of one of them and also ‘secondary isozymes’ due to post-translational modification.
