Genetic transformation may be defined as the transfer of heritable traits by introduction of naked DNA molecules into cells. Also, it may be defined as a genetic process through which the transfer of genetic information can occur (Mishra, 1985). Operationally transformation provides a method for self-cloning of fungal genes by complementation of mutant phenotypes (Johnson, 1985). This self-cloning approach is an attractive cloning method, particularly for A. nidulans and N. crassa which have many well characterised mutant alleles. The self-cloning technique is based on the usage of fungal genomic libraries constructed in plasmid or cosmid vectors to complement fungal strains after transformation. A complementary clone carrying the gene of interest can then be isolated by either rescuing in the bacterium E. coli (marker rescue) or by subcloning from positive pools of clones from plasmid or cosmid genomic libraries (Gems et. a i, 1991; Gems et. al., 1994; reviewed recently by Riach and Kinghorn, 1996).
Certain model organisms such as A. nidulans have a well characterised genetic linkage map (Figure 1.3), physical chromosome characterisation (physical or contiguous maps) and chromosome specific gene libraries constructed in plasmid, cosmid and phage vectors, and a number of already cloned genes. A more laborious procedure than self-cloning which takes advantages of these features is 'chromosome walking' : the isolation of overlapping cloned fragments, so as to proceed from a given point to any gene of interest.
Clones containing overlapping DNA fragments located progressively distal to the isolated gene are tested for phenotypic complementation of a mutation in the desired gene using standard transformation protocols. Overlapping clones can be organised into contiguous chromosomal regions (termed contigs) and used to construct physical genomic maps (contig maps). In A. nidulans,
cosmid libraries are available for each chromosome (Gibson et. al.,
1987; Wahl et. al., 1987; Brody et. al., 1991).
A recent transformation protocol is the method of transforming protoplasts by electroporation (the use of high voltage electric pulse to allow reversible permeabilisation of the cell membrane and uptake of DNA. The most common method for fungi transformation involves the preparation of protoplasts in the presence of an osmotic stabiliser (high salt or sugar concentration) in order to prevent cell lysis, followed by their successful regeneration (transformants grown on osmotically buffered selective medium) (see Figure 2.1, cloning strategy). Such transformation technology provides many ways of altering the genetic characteristics of fungi, and has been used for studying mechanisms that control growth, metabolism and development. Also, for the isolation and manipulation of genes of potential importance, ie. in medicine (genes required for antibiotics production), in industry (genes required for production of food additives such as citric acid).(Timberlake and Marshall, 1989; reviewed by Riach and Kinghorn, 1996).
Although A. nidulans itself, is not of direct or obvious commercial importance its properties makes it an attractive and useful model organism suitable for studying metabolic and developmental regulation. Also, it might act as a host for expression of genes cloned from other species e.g. as a host for expression of cloned genes from higher eukaryotes (Timberlake, 1980, Tilburn et. al., 1983; Arst, 1981, 1983; Johnstone, 1985).
Requirements For Transformation.
Three major requirements are needed for a successful transformation experiment, these are listed below:
1- A vector carrying a selectable marker, which after entry into the cell results in selective growth of only transformed cells.
2- Entrance of DNA into the cell and successful regeneration of the treated cells. This success depends on the viability of young protoplasts with competent cell membrane able to attach incoming DNA (ie. the vector) by rendering the cell wall permeable to the DNA. Selective conditions are subsequently applied to detect only the transformants that have incorporated and expressed the incoming DNA, and are capable to grow under selective conditions.
3- The expression and stable maintenance of transformed genetic material. Such maintenance can be achieved either by the autonomous replication, or by integration into the host chromosome (Johnstone, 1985; Gems et. al., 1991; reviewed by Riach and Kinghorn, 1996).
Factors Affecting Transformation Frequency.
The frequency of transformation in fungi is dependent on several factors listed below:
1. The nature of the recipient strain used in transformation.
2. The physiological state of the protoplasts, in which the preparation of viable transformable protoplasts depends on the age of cells, the choice of enzyme used for cell wall digestion, also the optimal timing for enzymatic digestion is crucial.
3. The nature of donor DNA and its purity. 4. The shape and size of the transforming DNA.
5. The ratio of the transforming DNA to that of the donor one.
6. Regeneration conditions. Also the ratio between the DNA and the protoplasts, and the density at which the protoplasts are plated on the selective medium after the treatment with the DNA is important (Mishra, 1985; reviewed by Saunders et. al., 1986; Fincham, 1989).
General Application Of Transformation.
Fungal transformation is a technique with applied and/or practical purposes:
1. The isolation of certain DNA sequence from a mixture of DNA molecules in the fungal cell ( as discussed above).
2. Insertion of an artificially synthesised nucleotide sequence, in order to examine its expression in fungal cells.
3. Modification of a desired nucleotide sequence in order to examine its effect on the expression of a gene.
4. For the commercial production of enzymes and proteins especially with elevated levels through the construction of hybrid plasmids. In this regard the gene that encode a desired enzyme or protein is linked to a fungal promoter and transcriptional activator carrying a signal sequence that finally terminates sequence and enables the cell to secrete the required product to the outside medium.
5. In biotechnology, e.g. for food industry, ethanol and organic acid fermentation’s, plant pathology, disposal of plant waste
6. For gene disruption perposes (reverse genetics) (Mishra, 1985; Fincham, 1989; Peberdy, 1991).
Molecular Cloning Techniques.
The DNA is normally partially digested with a restriction endonuclease into relatively small fragments (10 to 45 kb), is then inserted into a vector (plasmid, bacteriophage, or cosmid) carrying selectable marker (drug resistance) and cohesive ends which facilitates the ligation. Such hybrid plasmid vector may be used for transforming the host (e.g. E. co li, Aspergillus and yeast), where the presence of recombinant plasmid may be detected through the selectable marker. The presence of certain gene in this recombinant vector can be identified by a number of techniques, such as the com plem entation of mutants, immunological screening or hybridisation with a specific probe which could be DNA RNA or cDNA (reviewed by Mishra, 1985).
Vectors Used In Aspergillus.
The choice of vector for molecular cloning depends on both its capability of isolating and transferring the cloned gene via E. coli,
and on the nature of the gene fusion. Additionally, a vector should have a selectable marker and a unique cloning site, also it must be able to replicate in both fungus and bacteria. A substantial efforts have been put to develop a kind of vector from filamentous fungi which could replicate autonomously. The advantages of this type of vector not only they enhance the transformation frequencies but also they may be recoverd easily in E. coli. Recently a vector (and derivatives) have been developed in A. nidulans in which DNA insert (designated AM Al) which replicates autonomously with increased transformation frequency. Different kinds of vectors based on this have been used for gene cloning in fungi, their main characteristics are illustrated below (under plasmids section) (reviewed by Mishra,
1985; Saunders et. al., 1986; Riach and Kinghorn, 1996).
Features Of Plasmids Used In This Study.
These vectors were developed because of their small size, easy to replicate and possessed a defined number of cleavage sites for common restriction endonucleases. The most useful (i.e. transforming at high frequency) A. nidulans replicating plasmids are those designated ARpl, pDHG25, and pHelp, pDHG25 and pHelp, can be linearised at a unique site, thus directing the insertion of DNA fragment (Figure 1.5). ARpl; The replicating plasmid {Aspergillus
replicating plasmid), consists of pILJ16 with a 6.1 kb insert (ie. making total of 11.5 kb) (Johnstone et. al., 1985; Gems et al., 1991). The insert (AM Al: autonomously maintained in Aspergillus)
sequences came from A. nidulans . This vector exists in more than one copy in transformant cells. This DNA insert (i.e. AMAl)is responsible for autonomous replication of the plasmid vector, and has been used successfully for both increasing the transformation frequency and for the synthesis of ’instant gene banks’ when cotransformed with genomic DNA into an organism.. This vector is an autonomously replicating plasmid which transforms A. nidulans at frequencies much higher (up to 250 times) than pILJ16 (the parental plasmid). pDHG25; an autonomously replicating plasmid which is slightly smaller than ARpl (10.5 kb), and the outer 0.5 kb of AMAl on a Hin dlll fragment, also it has a unique Bam HI site which directs the insert to this site. It transforms at frequencies up to 1/40 of that of ARpl, but still higher (50 times) than the parental plasmid pILJ16.
pHelp: Markerless ARpl derivative (8 kb in size), having a unique
Bam HI site, in contrast, with the other two plasmids it does not carry the argB selective marker, but it has the AMAl sequence, which enhances the transformation efficiency. (Gems et. al., 1991; Gems et. al., 1993; reviewed by Mishra, 1985; Martinelli and Kinghorn, 1994; Riach and Kinghorn, 1996).