We have identified the promoter region of the GALIO gene (whose product is UDP-galactose epimerase) of Saccharomyces cerevisiae; this promoter mediates galactose induction of transcription in conjunction with the product of the GAL4 regulatory gene. This identification was achieved by excising a 365-base-pair fragment of GALIO leader DNA with.a GALlO proximal endpoint greater than 100 base pairs upstream of the transcriptional start site and substituting it in place of the-upstream, activation site of the CYCI (iso-l-cytochrome c) promoter [Guarente, L. & Ptashne, M. (1981) Proc. NatL Acad. Sci USA 78,[2199][2200][2201][2202][2203]. The.hybrid promoter is composed of DNA encoding CYCI mRNA start sites and the GAL segment upstream of these sites. This promoter is regulated in a manner analogous to GALIO; i.e., it is induced by galactose and responds to mutations in -the GAL4 and GAL80 regulatory loci. The activity of the hybrid promoter requires sequences in the region of the CYCI mRNA-start sites but does not require a precise spacing between these sequences and the GAL. segment. The transposed GAL segment appears not to contain sequences that mediate glucose repression. Thus, the~pic-ture of the GALIO promoter that emerges is one of an upstream activation site that responds to the GAL4 product plus galactose, and a region of transcription initiation thatmay contain sequences that mediate glucose repression. Experiments employing strains inducible (GAL80) or constitutive (galSO) for GALIO expression indicate that an additional component of glucose repression is inducer exclusion.Expression ofprokaryotic genes or eukaryotic genes transcribed by RNA polymerase II is regulated by DNA sequences that lie upstream of coding sequences. In the cases of the simian virus 40 early region (1, 2), the sea urchin histone H2A gene (3), or the yeast CYC1 gene (refs. 4 and 5) (unpublished data), these regulatory sequences lie in two regions, one in close proximity to where transcription initiates, and the other upstream of the initiation region. The CYCJ.gene, in particular, contains an upstream activation site (UASC) about 250 base pairs upstream of the startpoint of transcription that enhances expression about 50-fold (4).To further study the role of UAS regions, we have probed whether yeast genes other than CYCI contain such sites. The focus of this report is the GALlO gene of Saccharomyces cerevisiae. The product of GALJO (UDP-galactose epimerase), along with the products of GALl (galactokinase) and GAL7 (galactose-l-phosphate uridylyltransferase), forms the pathway for utilization of galactose as carbon source in S. cerevisiae. These three genes are closely linked in a cluster on chromosome II (6, 7) and are coordinately induced about 1,000-fold at the level of transcription by growth on galactose (8-10). This coordinate control is exercised by the constitutively synthesized protein products of the GAL4 and GAL80 genes, which are linked neither to each other nor to the structural gene cluster (11)(12)(13). The GAL...

The amino acid sequences of the repressor and cro proteins of phages lambda, 434 and P22 are homologous, especially in a region in which repressor and lambda cro have a similar alpha-helix-turn-alpha-helix secondary structure. Model-building studies indicate that this structure is important in DNA binding, and we suggest it may be a common feature of many DNa-binding proteins.

We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GALI-GALIO divergent promoter, 140 base pairs of GALIO coding sequence, and 87 base pairs of GAL) coding sequence. From this fragment, we constructed four pairs of GALI-lacZ and GAL1O-lacZ fusions on various types of yeast plasmid vectors. On each type of vector, the fused genes were induced by galactose and repressed by glucose. The response of a GALI-lacZ fusion to gaI4 and gal80 regulatory mutations was similar to the response of intact chromosomal GAL) and GALIO genes. A set of deletions that removed various portions of the GALIO regulatory sequences from a GAL1O-CYCI-lacZ fusion was constructed in vitro. These deletions defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for fullstrength galactose induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.The yeast Saccharomyces cerevisiae can grow on galactose as a sole carbon source. Galactose is transported into the cell by a specific galactose permease and is converted to glucose-i-phosphate by the sequential action of three enzymes, galactokinase, a-D-galactose-1-phosphate uridyltransferase, and uridine diphosphoglucose-4-epimerase (6, 32). These three enzymes are encoded by a tightly linked cluster of genes named GAL], GAL7, and GALIO, respectively (10). If cells are grown on glucose or glycerol and then switched to galactose, GAL], GAL7, and GALJO are coordinately induced at least 1,000-fold at the level of transcription (11,25,(59)(60)(61). This dramatic example of regulation in a genetically tractable organism provides an ideal system for studying a eucaryotic gene control mechanism.Expression of GAL], -7, and -10 is governed by at least two distinct regulatory circuits, a galactose-specific induction system (11) and a more general glucose repression system (1, 37). Much is known about galactose-specific induction (see reference 47 for review). Classical genetics and recent molecular studies have led to the following model. In the absence of galactose, GAL], GAL7, and GALIO are repressed by a negative regulatory protein encoded by the GAL80 gene (11). Recessive gaI80 mutations cause constitutive expression of GAL], GAL7, and GALIO, whereas dominant GAL80S mutations confer an uninducible phenotype (11)(12)(13)45). A positive regulatory protein, encoded by the GAL4 gene, is required for expression of GAL], GAL7, and GALIO, even in the absence of wild-type 25,35,36). Dominant GAL4C mutations cause constitutive expression of GAL], GAL7, and GAL10, whereas recessive gal4 mutations confer an uninducible phenotype (11,35,45). Both the GAL4 and GAL80 proteins are constitutively produced in low amounts (24, 28, 36 48).

An active fluorescent derivative of the actin-binding mushroom toxin phallacidin has been synthesized. Convenient methods were developed to stain actin cytoskeletal structures in living and fixed cultured animal cells and actively streaming algal cells. Actin binding specificity was demonstrated by competitive binding experiments and comparative staining of well-known structures. Large populations of living animal cells in culture were readily stained by using a relatively mild lysolecithin permeabilization procedure facilitated by the small molecular size of the label. Actin in animal cells was stained stress fibers, ruffles, the cellular geodome, and in diffuse appearing distributions apparently associated with the plasma membrane. Staining of actin cables in algae with nitrobenzoxadiazole (NBD)-phallacidin did not inhibit cytoplasmic streaming. NBD-phallacidin provides a convenient actin-specific fluorescent label for cellular cytoskeletal structures with promise for use in studies of actin dynamics in living systems.

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDB and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.