Purification of Fungi by Reporter Cloning

Reporter or reporter genes are nucleic acid sequences attached to a regulatory sequence gene

of interest in fungi.

The reporter gene are often used as an indication of whether the targeted (regulatory) gene

has been expressed in the cell. Reporter gene are easily assay proteins and are colour

changing in nature which can be easily identified by a small biochemical assay. Thus, by

analyzing the presence of reporter genes, a research can also ascertain how the gene of

interest (regulatory gene) is preforming in the cell. Reporter gene helps in ascertaining if the

gene of interest is present or absent in the cell.

Roles of a Reporter Gene in Fungi

1. The reporter gene helps in determining the strength of a promoter, because the more

the reporter gene is expressed is an indication that the promoter is very strong.

2. The reporter gene helps in the efficiency of gene delivery system.

3. The reporter gene helps in determining intracellular fate of a gene product.

4. Reporter gene help in the determining the success of molecular cloning effort.

Characteristics of a Reporter Gene in Fungi

1. The reporter gene must have low background activities in the target and absence of

any detrimental effect on metabolism.

2. The reporter gene should moderate stability in vivo so that down regulation of gene

expression as well as gene activation can be detected.

 Types of Reporter Gene

Most reporter genes are proteins that can be easily assay, they include:

1. CAT (chloramphenicol acetyltransferase)

Chloramphenicol acetyltransferase is a protein that transfers the radioactive acetyl

group to chloramphenicol. This can be detected by thin layer chromatography and

autoradiography.

2. GAL (β-galactosidase)

This protein hydrolyses colorless galactoside to yield colored products. It shows the

gene of interest is already incorporated into the cell.

3. GUS (β-glucuronidase)

This protein can hydrolyse the colorless glucuronide to yield colored products.

4. LUC (Lucifarase)

Lucifarase do not need a specific assay because any tissue that has luciferene gene,

the protein oxidise the luciferene and produces light.

5. GFP (Green Fluorescent Protein)

This assay do not need chemical whenever the target or fungi cell is subjected to UV

light it glows, which indicate is the regulatory gene is present or absent.