ersusually require an upstream activator sequence (UAS), an enhancer-like sequence located very far upstream (100 to 1000 bases) from (he transe! tion initiationsite. Because of the location of the UAS, most yeast ey Vectors contain a long, native “promoter sequence” (typically around 1 kb) ‘Two frequently used promoters are the upstream sequences for an aleahol dehydrogenase gene (ADH) and foratriose phosphate dehydrogenase gene (TDH3), ABH was thought to be expressed constitutively at a high level, and its use was popular at one time, However, we now know that this particular isozyme of alcohol dehydrogenase becomes repressed when the culture reaches high density, so its use has fallen off, (In-contrast, another isozyme ofalcohol dehydragenase, ADH2, becomes derepressed when glucose inthe medium becomes exhausted. The promoter for this enzyme is often used as a regulatable promoter, as we shall see.) Ifthe expression ofthe foreign protein inhibits the grawthoftheyeast cells, it becomes necessary to use regulatable promoters and to initiate expres- sion of the foreign genes only when the culture has reached a high den- sity. For example, the genes involved in galactose catabolism, GALJ, GAL7, and GALL0, have been extensively used as sources of regulatable promot- ers for cloned genes betause they are repressed fn the presence af glucose but are induced by the addition af galactose to the medium. the regula- tion of these genes involves the binding of a positive activator, GALS, to upstream sequences of GALI, GAL, and GALIO. Thus, if the recombinant DNA containing the latter genes exists in mmultiple copies in a cell and GAL4is expressed from a single copy of the gene on the chromosome, the GALS protein in the cell mighthecome exhausted by binding before all the recam~ binant genes are activated, However, this limitation can be removed if GALL isalso introduced into the Vector so that multiple eapies of G4L4 are present ina cell. A drawback of this system is that it tends ta increase the expres: sion of the cloned gene even in the absence of galactose, so it is dangerous the product is toxic to yeast cells. Other regulatable promoters that have been used include ADH2 (alcohol dehydrogenase regulated by ethanol and glucose) and PHOS (acid phosphatase, regulated by phosphate). Another attractive regulatable promoter is the one for CUP-1, which codes for met. allothionein, a Cu** -binding protein, and which is induced by the addition of metal ions such as Cu* or Zn** to the medium. Several systems thal are induced by elevated temperatures have been used successfully in the laba ratory, but it may be difficult to get the temperature to change fast enough ina large fermentation tank. Several hybrid promaters have also been used, ‘These contain (1) a UAS from a regulatable promoter for contralling the level of expression of the gene and (2) the TATA box region from a strong, constitutive promoter far increasing the maximal level af expression, Forexample, a hybrid promoter containing the JAS sequence of ADH? and the dawnstream sequences (con: taining the TATA box} from the TDH3 promoter has been effective in pra- ducing some foreign proteinsat levels sometimes exceeding 10% of the total yeast protein.