Investigators' Abstract) There are many genes in a living organism. With current technology it takes considerable time to investigate each one. Here the investigators propose to develop new reporter genes to facilitate these studies and to reduce the work involved in assaying gene expression and regulation. These reporters will allow multiple genes to be monitored simultaneously. In addition, they themselves have properties that may also open up new experimental possibilities in studies of protein structure and function. The availability of additional reporter genes will also facilitate studies of multiple gene and cell systems. Multiple genes in a cell, or genes in different cells, can be marked and followed independently. These new reporter genes will be especially useful for complex cell systems such as in developmental patterns. The type of indicator genes that the investigators seek primarily are hydrolyses like the Escherichia coli lacZ beta-galactosidase gene which can hydrolyze many substrates that have a common moiety such as a beta-galactosidase. The common moiety can be joined to many chemicals that can be detected easily by their color or light emission after they are liberated by the enzyme. Recently, the investigators have found stable versions of these types of enzymes, including beta-galactosidases, in thermophilic bacteria of the Thermus species. Currently the investigators are characterizing a thermo-beta-galactosidase enzyme whose gene tbg they have cloned from Thermus aquaticus, which is an easy to grow eubacteria. A unique advantage of enzymes from thermophilic bacteria is that they are often exceptionally stable and can function at temperatures well above any enzyme from a mammalian source. This allows them to be assayed even in the presence of an endogenous enzyme with the same activity. They can also be used as markers in a cell containing a mesophilic marker gene with the same activity. In particular, the investigators propose to screen for and clone potential new reporter genes from thermophilic or other microbial sources. These will be characterized and used to make vectors for basic molecular biological studies of genes and proteins in commonly studied organisms, including bacteria, yeast, and, with first priority, mammalian cells. Marker enzymes will be selected for their ease of use and ability to function under a wide range of conditions.
