Expression of Cloned Genes,Physical Analysis,Eukaryotic Genes,Bacterial Cell,Ribulose Bisphosphate Carboxylase,Charnay et al,Polypeptide

www.molecular-plant-biotechnology.info

Back to Home

Home >> Expression of Cloned Genes - Introduction >> Expression of Cloned Genes - Introduction

Back to Home

	Expression of Cloned Genes - In addition to purely physical analysis, investigations into the expression of cloned genes in host cells have been carried out. Many processes are involved in the expression of a gene and although some early experiments with hosts such as yeast and fungi were successful, transferred eukaryotic genes are often not expressed in their new host. Expression of cloned genes could be expected to yield valuable information regarding the processes involved and, from a biotechnological view point, expression of plant genes in bacteria may be of commercial significance.
For a cloned gene to be expressed in a bacterial cell, it has been found that it is necessary to place it under the control of an E. coli promoter. Many different plasmids have been constructed which allow insertion of a gene near a promoter. Some genes are inserted in such a way that the protein produced is actually fused to part of a bacterial protein, often β-galactosidase (e.g., β-endorphin), whilst others are inserted in such a way that the promoter is correctly placed for protein synthesis from the correct N-terminus. An example of this latter type would be maize gene coding for the large subunit of ribulose bisphosphate carboxylase.
Bacterial genes do not contain intervening sequences nor the machinery for removing them from the primary transcript. Eukaryotic genes containing introns will not give a functional mRNA molecule in bacteria even if transcribed efficiently. It is therefore, necessary to use cDNA clones of this type of gene for expression studies (e.g., sweet protein, thaumatin). A functional mRNA also depends on the coding sequence being in the correct reading frame if it has been fused to a bacterial coding sequence. Three vectors were constructed by Charnay et al. (1978) which allow cloning into the lac-Z gene of E. coli in all three possible reading frames relative to the initiation codon.
Similar vectors have been constructed by a number of other workers. If placed in the correct reading frame relative to the initiation codon and with a ribosome binding site present, the mRNA should be translated into a primary translation product. The problems do not end there, however; many proteins are modified in some way either by removal of the polypeptide or by addition of various groups. Signal sequences, which allow passage through membranes, need to be cloven and other modifications include glycosylation, adenylation and phosphorylation.
	While bacteria may be able to cleave signal sequences, in some cases glycosylation certainly does not occur. In the case of the sweet protein, thaumatin, neither the N- nor C-terminal extensions of the primary translation product were removed in E. coli. Likewise, the glutamine synthetase gene from Anabaena functions in E. coli but no adenylation of the enzyme occurs. Lack of these modifications may have certain consequences. If a functional protein is sought for some commercial application, lack of these modifications could be a serious problem. From an investigative point of view, the primary products may be very unstable.

If rapid degradation occurs, detection of the products would be difficult. Expression of a cloned gene requires correct functioning of a complete series of events.

A eukaryotic host, such as yeast, may have considerable advantages for some application as modification of primary translation products is known to occur in these organisms.

DNA Probes
Antisense RNA
Expression of Cloned Genes
In Situ Hybridization (ISH)
DNA Sequencing
Restriction Fragment Length Polymorphism (RFLP)
Polymerase Chain Reaction (PCR)