Cloned genes, controlling expression

Holton, T.A. et al., Cloning and expression of cytochrome P450 genes controlling flower colour. Nature, 366, 276, 1993. 

Expression of cloned genes. Once inside the microbe, the genes are then expressed by the normal protein synthesis equipment of the cell. The expression or production of the cloned material can be a significant problem as the promoters and operators may not be compatible with the control systems of these cells. Therefore, careful manipulation of these control sequences can enhance the production of the protein required. Similarly, the subsequent folding of the protein and/or excretion of the protein may well not be accomplished, and this is a symptom of a basic incompatibility between the host cell and the production of the foreign protein. 

Microarrays consist of at least many hundreds and usually many thousands of oligonuceolide sequences or cloned fragment or whole cDNAs printed onto a solid support (slide or membrane). Gene expression is analysed by labelling total mRNA from different treatments, such as pollutant exposed and control organism, with either a red or green fluorescent dye. Equal volumes of the labelled cDNA are mixed and hybridised to the DNA spots on the array slide. If the gene is expressed... 

The ideal internal control clone for microarray expression normalization would be derived from a gene whose expression level is invariant between the different states under investigation. This has been a problem for quantitative PCR studies for many years and is just as challenging for microarray. Unfortunately, such a clone is unlikely to be identified before carrying out an experiment. A pool of some 30-50 candidate normalization clones should therefore be contained within the array. 

However, complementation of the recessive mutation by the wild-type allele carried by one of the plasmid clones In the library allows a transformed mutant cell to grow Into a colony the plasmids bearing the wild-type allele can then be recovered from those cells. Because many of the proteins that regulate the cell cycle are highly conserved, human cDNAs cloned into yeast expression vectors often can complement yeast cell-cycle mutants, leading to the rapid Isolation of human genes encoding cell-cycle control proteins. 

Measures used to promote and control the expression of the cloned gene in the host cell during production should be described in detail. 

---