Genes, leaky

It is often difficult to distinguish a mutation, which causes a completely inactive protein to be produced instead of an enzyme, from an effective deletion of the gene, when neither protein nor enzyme is produced. If, however, a mutation causes a protein with weak enzymatic properties to be produced, the abnormal homozygote will be able to carry out the reaction, though at a lower rate than the normal the product of such a mutation is termed a leaky gene. ... 

In some families the enzymatic activity differs in different heterozygotes the gene itself can hardly change within these families, hence variability of enzymatic activity can occur without variation of the gene and leaky genes need not be invoked. 

An important feature in the mechanism of inheritance from the stand-point of our discussion lies in the existence of what have been inelegantly called "leaky genes" or less picturesquely "partial genetic blocks." Observation of a phenomenon of this sort was first made by Mitchell and Houlahanl in 1946 and was an outgrowth of the earlier pioneer studies on the genetics of Neurospora by Beadle and Tatum.2... 

Recipients of xylanase genes other than standard E. coli (a) B. subtilise (b) "leaky" mutant of E. coli (c) Pseudomonas putida. 

In 1991, several factors were demonstrated to affect the efficiency of relE controlled killing of E. colt when under lac promoter control (Knudsen Karlstrom, 1991)- Cells escaped suicide primarily because of the mutation rate and the leakiness of repression during normal growth. When relF was under lac JV5 promoter/operator control, the inactivation of suicide function through spontaneous mutation occurred at a frequency of <5 x 10 9. Knudsen has further theorized that if the number of suicide minus (mutant) bacteria in a culture can be kept at zero before induction, all cells will die. This can be achieved in two ways provide the suicide function in duplicate, and control the suicide gene expression so stringently, with a chromosomally located laclq gene, that a basal level of the suicide function to which cells can adapt will not be present (Knudsen et al., 1995). 

Figure 2.5 Conceptual examples of benefits and limitations of tightly regulated and leaky promoters. Therapeutic ranges for expressed genes are depicted by dotted lines. The left panel describes a situation in which the tightly-controlled promoter achieves regulated expression of therapeutic levels of gene product. The right panel illustrates an example where the leaky promoter is optional.

Most gene therapy applications require extravasation of the DNA carriers so that only relative small DNA complexes can pass through the blood vessels and interact directly with parenchymal cells after vascular administration [2]. Under pathophysiological conditions, the structure of the vasculature can change. This phenomenon - termed the enhanced permeation effect - has been utilized to passively target macromolecules to tumors, since blood vessels in tumors are relatively more leaky. 

Undeniably, not all targets are suited for HTS in recombinant microbes. In our own experience, we found that we were unable to complement a PFK strain of S. cerevisiae with the cDNA encoding H. contortus PFK, despite the fact that it was functionally expressed in E. coli (unpublished observations). Conversely, we initially attempted to construct an ODC screen using a strain of E. coli that was unable to synthesize poly amines [40], However, this strain required mutations in multiple genes in order to achieve the polyamine- phenotype, and proved to be too leaky for routine screening [40], The ODC- strain of S. cerevisiae was much better suited for HTS. 

Microfluidic structures used to retain beads might then fulfill the dual purpose of holding back particles while allowing samples and reagents to be delivered. A first type of device is presented in Fig. 7 in which microspheres are confined either in a microchamber bordered by a leaky wall (Fig. 7A) [27] or by a pillar made wall (Fig. 7B) [28]. Such systems were used to perform single nucleotide polymorphism (SNP) analysis of the codon 72 of the gene of the anti-cancer p53 protein. 

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