Nature Biotechnology pp 1041 - 1044

Wouldn’t it be nice to turn gene expression on and off rather like a light switch? Researchers have done just that, except that they have gone one step further and used light itself as the means of tripping genes on and off. In the October issue of Nature Biotechnology, researchers with the US Department of Agriculture (Albany, CA) and the University of Berkeley (Berkeley, CA) report a molecular switch that uses light to turn a gene on and off. Such a switch could potentially be used to determine the function of any gene, including an introduced transgene, in any cell, organism, or tissue that can be exposed to light.

Peter Quail and colleagues took advantage of the conformational properties of phytochrome, a chromoprotein that occurs naturally in plants. Phytochrome assumes two different shapes, Pr or Pfr, depending on whether it is exposed to red or far-red light. Only the Pfr form is capable of binding to another protein, PIF3. To exploit these properties to create a gene switch, Quail’s team engineered a yeast strain that produces two fusion proteins: one in which phytochrome is hooked onto a DNA-binding domain; and the other in which PIF3 is bound to a DNA-activation domain. Their idea was that gene expression would be induced only when the fusion proteins were in close proximity and binding upstream of the target gene. Sure enough, when they shone red light on the engineered yeast strain, the phytochrome in the first fusion protein changed from the Pr form to Pfr, which then was able to interact with PIF3 in the second fusion protein, bringing the DNA-binding domain and DNA activation domain together and inducing gene expression. Because of the reversible nature of the Pr/Pfr forms, Quail’s team could also turn off gene expression by placing the yeast in far red light and allowing the phytochrome to return to its natural Pr conformation, thus preventing the two fusion proteins from interacting.

The light triggered molecular switch has the advantage of allowing researchers to determine the function of a gene at different stages in a cell’s development, a benefit over other functional genomic methods, such as knockout technology. Also, the system is advantageous over other current molecular switches that need external chemical regulators because there are no uptake or toxicity problems.

Nature Biotechnology pp 1030 - 1034

Researchers have found a rapid way of finding the function of specific genes in rice, one of the world’s most vital staples. Previously, assigning function to rice genes was a rather hit and miss affair because researchers could only create mutants by randomly targeting genes through chemical treatment or the use of highly mobile pieces of DNA called transposons. In a paper in the October issue of Nature Biotechnology, Japanese researchers have developed a method for targeting genes specifically (rather than randomly) that works efficiently in rice.

To demonstrate their approach, Shigeru Iida and colleagues decided to target the native rice Waxy gene, which encodes an enzyme that influences the quality and quantity of rice grains. Using the knowledge that DNA sequences with the same sequence tend to swap around (recombine) in the genome, the Japanese team set out to determine whether introduction of an artificial DNA molecule with the same sequence as the Waxy gene could disrupt its structure and thereby produce visible effects on the targeted plants, allowing the investigators to infer gene function. Before they could do this, however, they had to make the recombination process much more efficient in rice. This was accomplished by making the DNA introduction method more efficient and refining the ways of selecting plants containing successfully targeted Waxy genes. Around 1 in 100 plants contained a disrupted Waxy gene, which is an unprecedented efficiency for gene targeting in higher plants. Because the selection approach is independent of the nature of the gene, the strategy could be more widely applied to other genes and has the potential to be used in many other crops.

Nature Biotechnology pp 999 - 1005

Most gene therapy protocols using adenovirus vectors currently offer a rather temporary solution to disease. Soon after the gene therapy vector has delivered its therapeutic gene to diseased cells, the gene is often lost or destroyed and the therapeutic benefit is lost. In a paper in the October issue of Nature Biotechnology, researchers at Stanford University School of Medicine have found a way of prolonging a therapeutic gene’s life by getting an adenovirus vector to integrate its genetic cargo into the target cells’ chromosomes. They accomplished this by hooking up their therapeutic gene to a piece of DNA (called a transposon) that naturally integrates into host chromosomes. By inserting the therapeutic gene into the host cell chromosome, they were able to achieve long-term gene expression.

To demonstrate their approach, Mark Kay and colleagues incorporated a transposon containing the sequence for human coagulation Factor IX (hFIX) into one adenoviral vector and the enzymes necessary for cutting and pasting the transposon into another vector. Systematic delivery of the viruses allowed stable integration of the transgene into mouse liver. Integration of the transgene was sufficient to maintain therapeutic levels of hFIX for more than six months in mice. Since integrated transgenes should persist for extended periods of time, these vectors may provide a new means to improve gene therapy.