Scientists who want to examine the properties and functions of specific proteins, as well as the activities of individual genes, must rely on chemical tags to manipulate and visualize them. This month's release of Cold Spring Harbor Protocols highlights methods for creating and detecting specific proteins, as well as for characterizing the activities of specific genes during embryonic development. Two of the methods are freely available online.

One of the freely available methods describes how to attach a small tag, called GST (glutathione- S -transferase), to a specific protein of interest. This is accomplished by growing bacteria that contain the DNA sequence for the GST tag adjacent to the gene for the protein of interest. When the bacteria grow, they express the "GST fusion protein," which consists of a GST tag attached to the desired protein. Using a chemical (glutathione) that binds specifically to GST, the GST fusion proteins can be easily sequestered and studied in more detail.

To test the activity of a certain gene during embryonic development, mice can be engineered to produce a "reporter" protein called -galactosidase, which functions as a proxy for the protein normally produced by the gene of interest. The second freely available method describes a strategy for staining transgenic mouse embryos engineered to express the -galactosidase protein. Once stained, the tissues with -galactosidase produce a brilliant blue pigment, allowing researchers to visualize where and when a gene of interest is expressed during embryonic development.

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In an effort to validate their earlier findings, the researchers have now generated mice carrying a version of eIF2a that cannot be phosphorylated. The mutant mice have lower levels of phosphorylated eIF2a and showed an improved talent for spatial learning in a water maze test.

In the test, the mice were trained to swim to a hidden platform. After several days of training, the altered mice were able to find the platform significantly faster than normal mice could, they reported.

"For example, if a person were reading a page of a textbook, it might take several times to memorize it," Costa-Mattioli said. "A human equivalent of these mice would get the information right away."

The mutant mice also performed better than normal mice in a "fear-conditioning" test. In that test, animals are put into a cage followed by a mild foot shock or are exposed to a tone paired with a foot shock. Their memory for the earlier bad experience is determined based on how much they freeze in response to the "scary" place or sound 24 hr later.

Importantly, the researchers also showed that treatment of animals with a small molecule that increases eIF2a's phosphorylation led to poorer performance on the memory tests.

"These data strengthen the idea that eIF2a phosphorylation is a key, bidirectional point of memory control," with the ability to turn long-term memory formation on and off, Costa-Mattioli said.

"Taken together, these data strongly support the notion that, under physiological conditions, a decrease in eIF2a phosphorylation constitutes a critical step for the activation of gene expression that leads to the long-term synaptic changes required for memory formation," the researchers concluded. "These findings also raise the interesting possibility that regulators of translation could serve as therapeutic targets for the improvement of memory, for instance in human disorders associated with memory loss."

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