Toshio Fukasawa

GAL11 protein, an auxiliary transcription activator for genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae.

Normal function of the GAL11 gene is required for maximum production of the enzymes encoded by GAL1, GAL7, and GAL10 (collectively termed GAL1,7,10) in Saccharomyces cerevisiae. Strains bearing a gal11 mutation synthesize these enzymes at 10 to 30% of the wild-type level in the induced state. In a DNA-RNA hybridization experiment, the gal11 effect was shown to be exerted at the transcription level. Yeast cells bearing the gal11 mutation were shown to grow on glycerol plus lactate more slowly than the wild type. We isolated recombinant plasmids carrying the GAL11 gene by complementation of the gal11 mutation. When the GAL11 locus was disrupted by insertion of the URA3 gene, the resulting yeast cells (gal11::URA3) exhibited phenotypes almost identical to those of the gal11 strains, with respect to both galactose utilization and growth on nonfermentable carbon sources. Deficiency of Gal4, the major transcription activator for GAL1,7,10, was epistatic over the gal11 defect. The Gal11 deficiency lowered the expression of GAL2 but not that of MEL1 or GAL80; expression of these genes is also known to be dependent on GAL4 function. We determined the nucleotide sequence of GAL11, which is predicted to encode a 107-kilodalton protein with stretches of polyglutamine and poly(glutamine-alanine). An alpha-helix-beta-turn-alpha-helix structure was found in a distal part of the predicted amino acid sequence. A possible role of the GAL11 product in the regulation of galactose-inducible genes is discussed.

Galactose-sensitive mutants of Salmonella II. Bacteriolysis induced by galactose

When the mutants of Salmonella lacking UDPGal-4-epimerase were grown in the presence of galactose, they exhibited marked bacteriolysis in ordinary medium and were convested to soheroplasts in hypertomic medium. Lysis was not observed in non-growing cells. Synthesis of cell wall does not seem to be inhibited by galactose at least up to 5–10 min before lysis, but cell wall synthesized in the presence of galactose had a sugar composition quite different from that formed in its absence. In the light of these findings, the mechanism of lysis was discussed, considering the metabolic consequences of the absence of epimerase.

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae

SummaryThe GAL4 gene positively regulating the expression of the gene cluster GAL7-GAL10-GAL1 in the yeast Saccharomyces cerevisiae was isolated for its ability to suppress a recessive mutation in that gene. When the isolated gene was incorporated into a multi-copy plasmid, the GAL cluster genes in the host chromosome partially escaped the normal control; a yeast that harbors the plasmid bearing the GAL4 gene synthesized the galactose-metabolizing enzymes encoded by the GAL cluster genes at a low but significant level in the absence of galactose. If the GAL7 gene was amplified along with GAL4 on the multi-copy plasmid, the constitutive synthesis of Gal-1-P uridylyl transferase encoded by GAL7 was further pronounced and the enzyme activity reached the level of the fully induced wild-type yeast. Such an escape synthesis of the GAL enzymes was not detected if GAL4 or both GAL4 and GAL7 were carried by a single-copy plasmid. The results suggest that the escape synthesis of GAL enzymes observed in the GAL4-amplified yeast was a consequence of overproduction of the GAL4 protein. The GAL80 gene negatively regulating the GAL cluster genes was also isolated, and when amplified together with GAL4, no escape synthesis of the GAL enzymes was observed, suggesting that the balanced synthesis of two regulatory proteins was essential to maintain the repressed state of the GAL cluster genes.

“Resistance transfer factor” an episome in enterobacteriaceae

Abstract The transfer of multiple drug resistance in Enterobacteriaceae was studied by conjugation and transduction. The resistance factors were found to be carried by an episome (“resistance transfer factor (RTF)”).

Regulation of expression of the galactose gene cluster in Saccharomyces cerevisiae - II. The isolation and dosage effect of the regulatory gene GAL80

SummaryThe galactose analogue 2-deoxygalactose was found to inhibit the growth of a mutant strain of Saccharomyces cerevisiae constitutively producing the set of galactose utilization enzymes. Based on this fact, the yeast GAL80 gene negatively regulating the expression of the genes encoding those enzymes was isolated for its ability to confer 2-deoxygalactose resistance on a strain carrying a recessive mutation in that gene. The GAL80 gene was located within a 3.0 kb fragment in the cloned DNA. When the isolated gene was incorporated into a multi-copy plasmid, the induced level of three enzymes encoded by the gene cluster GAL7-GAL10-GAL1 in the host chromosome was lowered. Such a gene dosage effect of GAL80 was further pronounced if sucrose, a sugar causing catabolite repression, was added to the growth medium. The ratio of the enzyme activity of the yeast bearing multiple copies of GAL80 to that of the yeast bearing its single copy significantly varied with the enzyme. From these results we suggest that the intracellular inducer interacts with the GAL80 product and that GAL80 molecules directly bind the GAL cluster genes with an affinity different from one gene to another.

A novel mutation that affects utilization of galactose in Saccharomyces cerevisiae.

SummaryA novel type of regulatory mutation for galactose metabolism in Saccharomyces cerevisiae is described. The mutation named gal11 was recessive, non-allelic to GAL4, GAL80, GAL2, or GAL3, and unlinked to the gene cluster of GAL1, GAL10, and GAL7. It caused a ‘coordinate’ reduction of galactokinase, galactose-1-P uridylyl transferase, and UDP-glucose 4-epimerase by a factor of more than 5, rendering the mutant cells galactose-nonfermenting. The effect of the mutation was manifested not only in cells grown on galactose but also in cells constitutively synthesizing the galactose-metabolizing enzymes.

Mechanism of host-controlled restriction of bacteriophage λ by R factors in Escherichia coli K12☆

Abstract In the host-controlled modification of phage λ by fi − R factors, N-3 and R-15, the infectivity of the unmodified λ DNA was specifically destroyed by the sonicated extracts from the restrictive, endonuclease I-less (endo I-less) mutant of Escherichia coli K12 carrying N-3 or R-15, to a greater extent than by the sonicates from the nonrestrictive, endo I-less bacteria. In the control experiments, the λ DNA modified by N-3 was inactivated to much lesser extents both by the sonicates from the restrictive, endo I-less bacteria and by those from the nonrestrictive, endo I-less bacteria. In all these systems of reactions, the phage DNA was hardly degraded into acid-soluble forms. The host specifically inactivated, unmodified λ DNA, however, was split fragmentally as was shown by the patterns of zone sedimentation, but the modified λ DNA was not. The activity which destroys the infectivity of the unmodified λ DNA resided totally in the spheroplasts of the restrictive bacteria.

Functional domains of a negative regulatory protein, GAL80, of Saccharomyces cerevisiae.

To study the functional domains of a transcriptional repressor encoded by the GAL80 gene of Saccharomyces cerevisiae, we constructed various deletion and insertion mutations in the GAL80 coding region and determined the ability of these mutations to repress synthesis of galactose-metabolizing enzymes as well as the capacity of the mutant proteins to respond to the inducer. Two regions, from amino acids 1 to 321 and from amino acids 341 to 423, in the total sequence of 435 amino acids were required for repression. The internal region from amino acids 321 to 340 played a role in the response to the inducer. The 12 amino acids at the carboxy terminus were dispensable for normal functioning of the GAL80 protein. Using indirect immunofluorescence and subcellular fractionation techniques, we also found that two distinct regions (amino acids 1 to 109 and 342 to 405) within the putative repression domain were capable of directing cytoplasmically synthesized Escherichia coli beta-galactosidase to the yeast nucleus. In addition, three gal80 mutations were mapped at amino acid residues 183, 298, and 310 in the domain required for repression. On the basis of these results, we suggest that the GAL80 protein consists of a repression domain located in two separate regions (amino acid residues 1 to 321 and 341 to 423) that are interrupted by an inducer interaction domain (residues 322 to 340) and two nuclear localization domains (1 to 109 and 342 to 405) that overlap the repression domains.

Autogenous regulation on the Saccharomyces cerevisiae regulatory gene GAL80

SummaryWe have suggested previously from Northern blot analysis that transcription of the negative regulatory gene GAL80 was controlled positively by another regulatory gene GALA, and negatively by GAL80 itself, in similar way to GAL1, GAL7 and GAL10 genes encoding galactosemetabolizing enzymes in Saccharomyces cerevisiae. To study further the controlled expression of GAL80, we have exploited the gene fusion technique. We constructed gene fusions consisting of 5′ fragments of GAL80 and a 5′ truncated lacZ of Escherichia coli, and introduced the GAL80‘-’lacZ fusions into wild-type yeast or various GALA or GAL80 mutants using multiple-copy or single-copy plasmid vectors. We then studied β-galactosidase activity in the resultant transformants under uninduced, induced or glucose-repressed conditions. Expression of the GAL80‘-’lacZ fusions was clearly under the control of Gal4/Gal80. Next we constructed GAL7‘-’lacZ fusions, whose upstream activating sequence (UAS) from GAL7 was replaced with a GAL80 fragment containing a UAS-like sequence located in the 5′ flanking region of GAL80. Synthesis of β-galactosidase directed by the hybrid genes was inducible by galactose exactly like the original GAL7‘-’lacZ fusion with a UAS from GAL7. Finally we constructed a GAL7-GAL80 hybrid gene, in which the entire 5′ flanking region was derived from GAL7. When the chromosomal GAL80 gene in wild-type yeast was replaced with the hybrid gene, the uninduced level, but not the induced level, of the GAL10-encoded enzyme (uridine diphosphoglucose-4-epimerase) was significantly increased.

Core promoter elements are essential as selective determinants for function of the yeast transcription factor GAL11

The GAL11 gene product, which copurifies with RNA polymerase II holoenzyme, is necessary for full expression of many, but not all, genes in yeast. Here we shows that the GAL11 dependence of a gene for expression is determined by the core promoter structure. In the GAL80 gene, a gal11 null mutation caused reduction of TATA‐dependent transcription, but exerted no effect on initiator‐mediated transcription. GAL11 stimulated TATA‐dependent transcription, but did not affect the TATA‐independent transcription in HIS4. GAL11 was also required for transcription mediated by a canonical TATA sequence but not by a nonconsensus TATA sequence of HIS3. These results suggest that GAL11 is specifically involved in the transcription machinery formed on the TATA element.