**1. Introduction**

66 Gel Electrophoresis – Advanced Techniques

Ye, W., Tan, J., Liu, X., Lin, S., Pan, J., Li, D., Yang, H., 2011. Temporal variability of

determined by DGGE and real-time PCR. *Harmful Algae*, 10: 472-479.

cyanobacterial populations in the water and sediment samples of Lake Taihu as

Woese, C.R., 1987. Bacterial evolution. Microbiology Reviews, 51, 221–271.

Several articles were written from the beginning of the fifties about the presence of plant enzymes in multiple forms. The major discussion was questioning whether these forms are artifacts that rose during the purification or not. To show that these forms are not artifacts in 1952 Jermyn divided his original peroxidase juice into two parts by acidic precipitation. The precipitate contained the A and B, while the supernatant the C and D points. Two components were found in the purified peroxidase solution; one migrated to the anode, the other to the cathode (Jermyn and Thomas, 1954).

The first major step for the starting up of isozyme analysis was the development of starch gel electrophoresis by Smithies (1955). The second major step was the demonstration of the direct visualization of isozymes in the stach gel by specific histochemical stains by Hunter and Markert in 1957 (McMillin in Tanksley and Orton, 1983).

The term isozyme was formed by Market and Moller (1959), using this word for different molecular forms of enzymes with the same substrate specifity.

Proteins - as the primary products of structural genes - are very alluring for the direct genetic studies. Variation in the DNA coding sequences frequently (but not all the cases) causes variation in the primary conformation of the proteins. In un-natural environments the detection of this variation is very difficult, because in such conditions the base of the separation is only the size of the protein (molecular weight). In natural environments the change of a single amino acid can detectably modify the migration. The extraction from a single tissue can contain a lot of proteins, which - in the case of non-specific (e.g. Comassie blue) staining - can result in a complex pattern, that makes it difficult to identify the homolog (allelic) and non-homolog enzymes. This problem can be solved by the application of enzyme-specific staining after the electrophoresis (Shields et al. in Tanksley and Orton, 1983).

The analysis of isozymes and their functions is the subject of functional genomics. The study of the gene expression in the level of RNA and proteins can give answers to a lot of open questions (Bernardi, 2004).

Gel Electrophoresis of Grapevine (*Vitis vinifera* L.) Isozymes - A Review 69

of a certain species, whereas multiple loci are common to all members of a species

The most probable reason for the presence of multilocus enzyme forms is the gene duplication. The gene duplication — the multiplication of genes in the genom —can come into existence by, for example, not equal crossing over. The frequency of mutation of various structural genes can be different, as a result of which some genes only rarely present in different allelic variant, as more alleles present in the population of other isoforms. This

For the formation of multilocus isoforms a different evolutionary way can be imagined. It is probable, that the variation of the structural genes of originally different enzymes can cause

Enzymes with variable substrates generally show higher variability itseves (catechol oxidase, acid phosphatase, peroxidase, esterase), but the amount of allozymic polymorphism is an increasing function of environmental variation. "Observations on natural pupulatians are cited which substantiate the claim that allozymic polymorphism is primarily due to selection acting on environmental variation in gene function. …a large portion of the observed allozymic variation is due to a rather specific type of phenomenon:

Enzymes with a single, special substrate show lower variability (glucose phosphate isomerase, phosphoglucomutase, glutamate-oxalacetate transaminase, glucose-6-phosphate transaminase etc.), but the banding patterns are less affected by the environment (Gillespie

Isoenzymes can be separated by electrophoresis or isoelectric focusing. The isozymes under given proper circumstances — show peculiar patterns in the gel, which are called

Electrophoresis is a type of chromathography. The power for the separation of proteins is the difference in voltage between the two ends of the gel. The movement of proteins in the electric field is effected by their weight, shape and charge (Smith, 1960; Bálint and Bíró in

The gel for the separation can be made from starch, agarose or acrilamide (Fig 2.). A standardized method of starch gel electrophoresis is used by UPOV (1996) for the analysis of

Advantages of the starch gel are that it is non-toxic, and more isozymes can be analysed by the slicing of a thick gel. More recently polyacrylamide gels are used because of their larger resolution. The porous structure of poliacrilamide gel is formed through a process of polymerization of acrylamide (CH2=CH-CO-NH2) and bis-acrylamide (CH2=CH-CO-NH-CH2-NH-CO-CH=CH2). As a result of polimerisation a colourless, diaphanous, flexible, and consistent gel arises, which is resistant to scalding or chilling. The density, viscosity and size of poles are determined by the concentration of acrylamide and bis-acrylamide. (Hajósné

difference can be accepted as the evidence of a separate locus.

the formation of similar catalytic functions. (H. Nagy, 1999).

substrate variability" (Gillespie and Langley, 1974).

and Kojima, 1968; Gillespie and Langley, 1974).

zymogramm (Hunter and Markert 1957).

Bíró, 1989).

**3. Separation of isozymes by gel electrophoresis** 

identity of plant cultivars by isozyme analysis (Baum, 1986).

Novák és Stefanovitsné Bányai in Hajósné Novák, 1999).

(Markert, 1975.).

Fig. 1. A photograph of the result obtained by vertical starch-gel electrophoresis (approx. 19 hr. at 5v/cm.) with serum samples from six healthy individuals. Only the section of gel from the sample slots to the albumin is included in the photograph. Samples 1 and 2 are from female identical twins, 45 years old. Samples 3-5 are from 9-year-old female non-identical quadruplets and sample 6 is from their mother. (Smithies, 1958)
