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Transcription and mRNA export are linked processes. However, the molecular mechanisms of this coordination are not clear. Sus1 (hENY2) participates in this coordination as part of two protein complexes: SAGA, a transcriptional co-activator; TREX-2, which functions in mRNA biogenesis and export. Here, we investigate the coordinated action of SAGA and TREX-2 required for gene expression. We demonstrate that TREX-2 subunit Sem1 also participates in transcription activation. Like Sus1, Sem1 is required for the induction of ARG1 and GAL1, these being SAGA-regulated genes. Chromatin immunoprecipitations show that proper recruitment of certain SAGA subunits to the GAL1 promoter depends on Sem1. Notably, both in vivo and in vitro analyses reveal that Sem1 influences SAGA-dependent histone H2B deubiquitylation. Most of these phenotypes are also found to depend on another TREX-2 subunit, Thp1. These results unveil a new role for Sem1 in the activation of the SAGA-dependent gene GAL1 and influencing H2B deubiquitylation. Our work provides insights into a novel functional relationship between Sem1 and the SAGA complex.

Galactokinases (GALK) have attracted significant research attention for their potential application in the enzymatic synthesis of unique sugar phosphates. The galactokinase (GalKSpe4) cloned from Streptococcus pneumoniae TIGR4 had a temperature optimum of 45°C, and a pH optimum of 8.0. The substrate specificity and kinetics studies revealed that GalKSpe4 had moderate activity toward glucose, in contrast with very low or no activity observed in other previously reported GALKs. Most interestingly, GalKSpe4 exhibited activity for GalNAc, which had never been recorded in other GALKs found by now. This is the first time to report that bacterial GALK can recognize GalNAc., (Copyright © 2011. Published by Elsevier Ltd.)

Rtt109p, a histone acetyltransferase, associates with active genes and acetylates lysine 56 on histone H3 in Saccharomyces cerevisiae. However, the functional role of Rtt109p or H3 Lys(56) acetylation in chromatin assembly/disassembly (and hence gene expression) immediately switching transcription on or off has not been clearly elucidated in vivo. Here, we show that Rtt109p promotes the eviction of histone H3 from a fast inducible yeast gene, GAL1, following transcriptional initiation via histone H3 Lys(56) acetylation. Conversely, the deposition of histone H3 to GAL1 is significantly decreased in the presence of Rtt109p following transcriptional termination. Intriguingly, we also find that the deposition of histone H2B on preexisting non-acetylated histone H3 Lys(56) at GAL1 in Δrtt109 is significantly increased independently of histone H3 deposition immediately following transcriptional termination subsequent to a short induction. Consistently, histone H2B is not efficiently evicted from GAL1 in the absence of Rtt109p immediately following transcriptional induction. Furthermore, we show that the stimulated eviction or reduced deposition of histones by Rtt109p promotes the association of RNA polymerase II with GAL1 and hence the synthesis of GAL1 mRNA. These results, taken together, support the fact that Rtt109p regulates the deposition/eviction of histone H2B in addition to its role in stimulating histone H3 eviction, thus providing insight into chromatin assembly/disassembly and hence gene expression in vivo.

The aim of this work was to construct a novel food-grade industrial arming yeast displaying beta-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The beta-1,3-1,4-glucanase gene from Bacillus subtilis was fused to alpha-agglutinin and expressed under the control of the GAL1 promoter. alpha-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the alpha-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of beta-1,3-1,4-glucanase. The analysis of beta-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that beta-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed beta-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of beta-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.

Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of alpha-D-galactose to alpha-D-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with alpha-D-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 A resolution for the apo form and to 1.7 A for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 A, beta = 109.8 degrees. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.

Genes for the enzymes that metabolize galactose in Saccharomyces cerevisiae are strongly induced by galactose and tightly repressed by glucose. Because glucose also represses mitochondrial activity, we examined if derepression of the GAL1 galactokinase gene requires physiologically active mitochondria. The effect of mitochondria on the expression of GAL1 was analyzed by a novel approach in which the activity of the organelles was altered by functional expression of URF13, a mitochondrial protein unique to the Texas-type cytoplasmic male sterility phenotype in maize. Mitochondrial targeting and functional expression of the URF13 protein in yeast result in a decrease of the mitochondrial membrane potential similar to those observed in cells treated with mitochondrial inhibitors such as antimycin A or sodium azide. Activation of URF13 in galactose-induced cells results in the inhibition of GAL1 expression in the absence of repressing concentrations of glucose. Our data reveal the existence of a regulatory pathway that connects the derepression of the GAL1 gene with mitochondrial activity.

Carbon catabolite repression by the CreA-transcriptional repressor is widespread in filamentous fungi, but the mechanism by which glucose triggers carbon catabolite repression is still poorly understood. We investigated the hypothesis that the growth rate on glucose may control CreA-dependent carbon catabolite repression by using glucose-limited chemostat cultures and the intracellular beta-galactosidase activity of Aspergillus nidulans, which is repressed by glucose, as a model system. Chemostat cultures at four different dilution rates (D = 0.095, 0.068, 0.045 and 0.015 h-1) showed that formation of beta-galactosidase activity is repressed at the two highest Ds, but increasingly derepressed at the lower Ds, the activity at 0.015 h-1 equalling that in derepressed batch cultures. Chemostat cultures with the carbon catabolite derepressed A. nidulans mutant strain creADelta4 revealed a dilution-rate independent constant beta-galactosidase activity of the same range as that found in the wild-type strain at D = 0.015 h-1. Two other enzymes--isocitrate lyase, which is almost absent on glucose due to a CreA-independent mechanism; and galactokinase, which is formed constitutively and independent of CreA--were measured as controls. They were formed at constant activity at each dilution rate, both in the wild-type strain as well as in the carbon catabolite derepressed mutant strain. We conclude that the growth rate on glucose is a determinant of carbon catabolite repression in A. nidulans, and that below a certain growth rate carbon catabolite derepression occurs.

The numerous changes in metabolic pathways that accompany liver development entail associated changes in gene expression. Egr-1 is a zinc-finger transcription factor that regulates genes involved in cellular growth, differentiation, stress response, and apoptosis in many cell types. Egr-1 is induced in liver regeneration in rodents, but its role in normal hepatocyte function has not been characterized. We examined the developmental expression of Egr-1 in mouse liver and found that its expression increased during the suckling period. In screening the sequences of the genes involved in lactose assimilation, we found that the galactokinase gene Glk contains four potential Egr-1 binding sites in its proximal promoter. A minimal promoter of 155 nucleotides encompassing the four Egr-1 sites exhibited activity in hepatoma cell lines by transient transfection assays. Moreover, co-transfection of an Egr-1 expression plasmid increased promoter activity. Finally, mutations introduced into three of the four Egr-1 binding sites decreased activity, whereas mutation of the remaining site increased promoter activity. These data tie Egr-1 and galactokinase together in a developmentally regulated chain to prepare the neonate for suckling.

Galactokinase catalyses the conversion of galactose to galactose-1-phosphate as the first step in the Leloir pathway, a metabolic route that eventually enables the degradation of galactose via the glycolytic pathway. Galactokinases have been isolated from a wide range of prokaryotic and eukaryotic organisms and the enzyme has been identified as a member of the GHMP kinase (galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase) superfamily. Pyrococcus furiosus galactokinase was cloned, expressed in Escherichia coli, purified and crystallized using the hanging-drop method of vapour diffusion with ammonium sulfate as the precipitant. The crystals belong to the space group C222(1), with more than eight subunits in the asymmetric unit and with approximate unit-cell parameters a = 211.7, b = 355.4, c = 165.5 A, alpha = beta = gamma = 90 degrees. The crystals diffract X-rays to 2.9 A resolution on a synchrotron-radiation source. Determination of the structure will provide insights into the molecular basis of substrate recognition and catalysis of this enzyme, for which no structures are currently available.

The SAGA complex of Saccharomyces cerevisiae is required for the transcription of many RNA polymerase II-dependent genes. Previous studies have demonstrated that SAGA possesses histone acetyltransferase activity, catalyzed by the SAGA component Gcn5. However, the transcription of many genes, although SAGA dependent, is Gcn5 independent, suggesting the existence of distinct SAGA activities. We have studied the in vivo role of two other SAGA components, Spt3 and Spt20, at the well-characterized GAL1 promoter. Our results demonstrate that both Spt3 and Spt20 are required for the binding of TATA-binding protein but not of the activator Gal4 and that this role is Gcn5 independent. These results suggest a coactivator role for Spt3 and Spt20 in the recruitment of TBP.

The contribution of the dosage of target enzyme P-450 14alpha-demethylase (14alphaDM) to fluconazole resistance in both Candida albicans and Saccharomyces cerevisiae remains unclear. Here, we show that overexpression of Saccharomyces P-450 14alphaDM in S. cerevisiae, under the control of the regulatable promoter GAL1, results in azole resistance.

It was found that Mycobacterium smegmatis is unable to utilize galactose as the sole carbon source because the sugar alone cannot induce galactokinase. However, galactokinase was induced by glutamate alone, and was further stimulated by galactose. Rifampicin completely inhibited the glutamate-mediated expression of galK in both the absence and presence of galactose. Extracellular cAMP stimulated the expression of the enzyme only in the presence of glutamate plus galactose. The galK gene from M. smegmatis, including its upstream promoter region, was cloned in a plasmid in Escherichia coli. The expression of kinase from these clones in E. coli was dependent on cAMP and its receptor protein (CRP). The expression of UDP-galactose 4-epimerase was constitutive. This and other evidence suggests that the galK gene is not linked to galT and galE in the mycobacterial genome. In a glutamate-independent galactose-utilizing mutant (gin-1 mutant) of M. smegmatis, galK was expressed in the absence of both galactose and glutamate, while in the presence of galactose this expression was increased twofold in the absence of glutamate and fourfold in its presence. Extracellularly added cAMP reduced the expression of the enzyme in the presence of galactose plus glutamate nearly to the basal level. It is proposed that in M. smegmatis the galK gene is expressed from two different promoters; the expression from one promoter is dependent on glutamate but not on galactose and cAMP, while that from the other requires all three components. The role of galactose is possibly to derepress the latter promoter.

A cDNA clone encoding Arabidopsis thaliana galactokinase was fortuitously isolated during the course of a screen for plant homologues of a Saccharomyces cerevisiae peroxisome assembly gene, PAS9. Clones were sought which restored the ability of pas9 cells to grow on oleate as a sole carbon source, as oleate metabolism requires peroxisomal beta-oxidation and therefore functional peroxisomes. Subsequent experiments showed that high level expression of the galactokinase cDNA did not complement the peroxisomal assembly defect, but instead permitted the cells to grow on agar plates in the absence of an external carbon source. Agar plates were shown to contain a small amount of galactose released from the agar as a result of autoclaving. The galactokinase clone was shown to be functional, as it could complement a S. cerevisiae galactokinase mutant. Galactokinase is a single copy gene in Arabidopsis, which has been designated AGK1, and is expressed in all the major organs of the plant.

Inversion of the ihf site in the promoter region of the early promoter of bacteriophage Mu did not influence the integration host factor (IHF)-mediated functions. IHF bound to this inverted site could counteract H-NS-mediated repression, directly activate transcription, and support lytic growth of bacteriophage Mu. This implies that the IHF heterodimer and its asymmetrical binding site form a functionally symmetrical complex.

In Saccharomyces cerevisiae, there are a large number of genes (HXT1-HXT17/SNF3/RGT2) encoding putative hexose transporters which, together with a galactose permease gene (GAL2), belong to a superfamily of monosaccharide facilitator genes. We have performed a systematic analysis of the HXT1-7 and GAL2 genes and their function in hexose transport. Glucose uptake was below the detection level in the hxt1-7 null strain growing on maltose. Determination of the kinetic parameters of individual hexose transporter-related proteins (Hxtp) expressed in the hxt null background revealed Hxt1p and Hxt3p as low-affinity transporters (Km(glucose) = 50-100 mM), Hxt2p and Hxt4p as moderately low in affinity (Km(glucose) about 10 mM), and Hxt6p, Hxt7p as well as Gal2p as high-affinity transporters (Km(glucosse) = 1-2 mM). However, Hxt2p kinetics in cells grown on low glucose concentrations showed a high-affinity (Km = 1.5 mM) and a low-affinity component (Km = 60 mM). Furthermore, we investigated the involvement of glucose transport in glucose signalling. Glucose repression of MAL2, SUC2 and GAL1 was not dependent on a specific transporter but, instead, the strength of the repression signal was dependent on the level of expression, the properties of the individual transporters and the kind of sugar transported. The strength of the glucose repression signal correlated with the glucose consumption rates in the different strains, indicating that glucose transport limits the provision of a triggering signal rather then being directly involved in the triggering mechanism.

The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae. Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL. Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression. This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p. The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose. When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose. Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p. Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence. Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids.

The yeast Snf1p kinase is required for normal expression of many genes involved in utilization of non-glucose carbon. Snf1p is known to associate with several proteins. One is Sip1p, a protein that becomes phosphorylated in the presence of Snf1p and thus is a candidate Snf1p kinase substrate. We have isolated the SIP1 gene as a multicopy suppressor of the gal83-associated defect in glucose repression of GAL gene expression. Multicopy SIP1 also suppressed the gal82-associated defect in glucose repression, suggesting that SIP1, GAL83 and GAL82 function interdependently. Multicopy SIP1 gene reduces GAL1, GAL2, GAL7 and GAL10 gene expression three- to fourfold in cells grown in the presence of glucose but has no effect in cells grown on nonrepressing carbon. Sip1-deletion cells exhibited a two- to threefold increase in GAL gene expression compared to wild-type cells when grown on glucose. These studies show that SIP1 is a catabolite repression-specific negative regulator of GAL gene expression. Northern analysis revealed two SIP1 transcripts whose relative abundance changed with carbon source. Western blots revealed that Sip1p abundance is not markedly affected by carbon source, suggesting that Sip1p may be regulated post-translationally.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.

In Salmonella typhimurium the metE and metR promoters overlap and are divergently transcribed. Three tandem repeats of an 8 bp sequence defined previously as the metE operator site for MetJ-mediated repression also overlap the -35 region of the metR promoter. Starting with a metE-lacZ.metR-galK double gene fusion, site-directed mutagenesis was used to change nucleotides in each of the repeat units from the consensus sequence. Each mutation, along with the wild-type metE-lacZ.metR-galK gene fusion, was cloned into phage lambda gt2. Regulation of the metE and metR genes was examined by measuring beta-galactosidase and galactokinase levels in Escherichia coli strains lysogenized with phage carrying the wild-type and mutant fusions. Mutations in each of the 8 bp repeat units disrupt MetJ-mediated repression for both the metE-lacZ and metR-galK gene fusions, suggesting that the metE and metR genes share a common operator site for the MetJ repressor.

In Escherichia coli, transcription of the heat shock genes is regulated by sigma 32, the alternative sigma factor directing RNA polymerase to heat shock promoters. sigma 32, encoded by rpoH (htpR), is normally present in limiting amounts in cells. Upon temperature upshift, the amount of sigma 32 transiently increases, resulting in the transient increase in transcription of the heat shock genes known as the heat shock response. Strains carrying the rpoH165 nonsense mutation and supC(Ts), a temperature-sensitive suppressor tRNA, do not exhibit a heat shock response. This defect is suppressed by rpoD800, a mutation in the gene encoding sigma 70. We have determined the mechanism of suppression. In contrast to wild-type strains, the level of sigma 32 and the level of transcription of heat shock genes remain relatively constant in an rpoH165 rpoD800 strain after a temperature upshift. Instead, the heat shock response in this strain results from an approximately fivefold decrease in the cellular transcription carried out by the RNA polymerase holoenzyme containing mutant RpoD800 sigma 70 coupled with an overall increase in the translational efficiency of all mRNA species.

Escherichia coli TG1 (pHRW500) permanently expressed the human interferon alpha 1 gene (ifn alpha 1) directed by the tryptophan promoter (trpP.O) during continuous and fed-batch cultivation with a limited supply of glucose. The expression of ifn alpha 1 could be improved after insertion of the catabolite activator region (cap) upstream to trpP.O during cultivation of the modified E. coli TG1(pHRW500cap) in glucose-controlled continuous and fed-batch cultures. The cap-mediated stimulatory effect on the expression of cap-trpP.O-ifn alpha 1 increased with decreasing dilution rate. These results are in line with the increase in the level of cAMP with declining dilution rate and the well-known positive effects of cAMP-catabolite gene activator protein (CAP) at the transcriptional level. In addition, expression of the galactokinase gene (trpP.O-galK) in E. coli TG1(pDR720) could be improved in the same way with cap-trpP.O-galK in E. coli TG1(pDR720cap). Determinations of plasmid copy numbers, cellular amounts of galactokinase-mRNA, activity of galactokinase (AGalK) and the concentration of galactokinase at various dilution rates (D) strengthen the conclusion that the increase in AGalK with decreasing D was indeed due to the cap-mediated enhancement of transcription of the galK gene. We suggest that expression of other recombinant genes directed by various promoters that allow permanent transcription during growth with limited glucose supply in chemostat and fed-batch fermentors can be improved by appropriate insertion of the cap region.

We have analyzed a GAL1 mutant (gal1-r strain) of the yeast Kluyveromyces lactis which lacks the induction of beta-galactosidase and the enzymes of the Leloir pathway in the presence of galactose. The data show that the K. lactis GAL1 gene product has, in addition to galactokinase activity, a function required for induction of the lactose system. This regulatory function is not dependent on galactokinase activity, as it is still present in a galactokinase-negative mutant (gal1-209). Complementation studies in Saccharomyces cervisiae show that K. lactis GAL1 and gal1-209, but not gal1-r, complement the gal3 mutation. We conclude that the regulatory function of GAL1 in K. lactis soon after induction is similar to the function of GAL3 in S. cerevisiae.

The nusA11 mutation causes reduced transcription termination and temperature-sensitive growth of Escherichia coli. Suppressor mutations that restored growth of nusA11 mutant cells were isolated and named sna mutations. The intergenic suppressor mutation sna-10 was located in the rpoC gene at 90 min, which encodes the beta' subunit of RNA polymerase. sna-10 complemented the defect in tR1 termination caused by nusA11 and by itself stimulated termination of transcription at the lambda tR1 terminator. sna-10 is specific to the nusA11 allele and unable to suppress cold-sensitive growth of the nusA10 mutant. nusA10 carried two base substitutions at positions 311 and 634, causing two amino acid changes from the wild-type sequence. During these studies, we found three -1 frameshift errors in the wild-type nusA sequence; the correct sequence was confirmed by the peptide sequence and gene fusion analyses. The revised sequence revealed that nusA1 and nusA11 are located in an arginine-rich peptide region and substitute arginine and aspartate for leucine 183 and glycine 181, respectively. The intragenic suppressor study indicated that the nusA11 mutation can be suppressed by changing the mutated aspartate 181 to alanine or changing aspartate 84 to tyrosine.

A segment of Escherichia coli bacteriophage 21 DNA encoding the late-gene regulator, Q21, and the late-gene leader RNA segment was sequenced; its structure is similar to those of the related phages lambda and 82. The leader RNA is about 45 nucleotides long and consists essentially entirely of sequences encoding the p-independent terminator that is the putative target of the antitermination activity of Q21. Like the corresponding regions of lambda and 82, the 21 late-gene promoter segment encodes an early transcription pause in vitro, at about nucleotide 18, during which Q21 presumably acts to modify RNA polymerase. The 21 Q gene, cloned in isolation, is active on the late-gene leader segment in trans, and its purified product is active as an antiterminator in vitro; Q21 represents a third late-gene antiterminator, in addition to those of lambda and 82. There is little evident similarity in the primary sequences of the three Q genes.

We have used a prokaryotic promoter-identification vector, pKO-1, to isolate rat mitochondrial DNA (mtDNA) sequences that can act as bacterial transcription promoters. Three putative promoter-containing clones that hydridized to mtDNA probes were identified. The strength of the promoters was quantitated by measuring galactokinase activity. The three promoters mapped to three distinct regions of the mtDNA - one within the 5' half of the 16S rRNA gene, one within the ATPase subunit 6 gene, and the last at the carboxy terminal end of the cytochrome oxidase subunit I gene.

The loss of expression of the enzyme O6-methylguanine-DNA methyltransferase (the Mex- phenotype), which often results from cellular transformation, confers hypersensitivity to alkylating agents. We have observed two unrelated examples in which human cell lines have undergone a spontaneous alteration in their Mex phenotype during propagation in vitro. The change was reversible and was not the result of mutation. In both cases a loss of methyltransferase expression was accompanied by a simultaneous loss of expression of two metabolically unrelated enzymes: thymidine kinase and galactokinase. "Reversion" to methyltransferase expression was accompanied by simultaneous reexpression of both kinase activities. A third example of this coordinate gene regulation was seen with the Burkitt's lymphoma cell line Raji which expresses methyltransferase, thymidine kinase, and galactokinase at high levels. A thymidine kinase- Raji cell line derived by bromodeoxyuridine mutagenesis that is also Mex- was found to be galactokinase-. It appears that methyltransferase expression may in some instances be coordinately regulated with the tk and glk loci which are closely linked on human chromosome 17.

A collection of variant plasmids expressing either Escherichia coli galactokinase or human serum albumin under the control of several E. coli trp promoter derivatives were constructed and studied for both efficiency of expression and regulation by tryptophan. Several variables, including the length of the upstream region, tandem duplications of a core promoter, and the insertion of the trp repressor trpR gene onto the expression vector, were studied. It is shown that derivatives containing sequences upstream from the -35 region or multiple copies of the trp promoter produce twofold higher levels of protein than plasmids with a minimal trp promoter truncated at -40. We show that the expression of a heterologous protein such as albumin can be significantly improved (13% vs. 7% of total proteins) if both the upstream trp promoter region, which enhances promoter strength, and an intact trpR gene, are included on the plasmids.

The steady-state level attained for any protein in mammalian cells is in part determined by its steady-state level of mRNA. Sequence information in and around the 3' end of an RNA which is involved in specifying and regulating polyadenylation [poly(A)] may have important consequences on mRNA levels, and ultimately on expression of the protein product. In this report we compare the effects on gene expression which result from placing several different poly(A) regions, or no poly(A) region, downstream from a marker gene (galactokinase or galK) that can be readily assayed in mammalian cells. Our results demonstrate that the presence of a poly(A) region is important for efficient gene expression and that the use of the poly(A) region of bovine growth hormone (bGH) reproducibly results in three times higher expression than that of SV40 early or human collagen poly(A) regions. We further demonstrate that changing the promoter region on these chimeric transcription units does not change the effect of the poly(A) region. Neither does changing the assay gene, since comparison of the same poly(A) regions behind another marker gene (xanthine-guanine phosphoribosyl transferase or xgprt) leads to identical differences in expression. When we examine the levels of poly(A)+ RNA that result from each transcription unit, we find that they correlate precisely with the gene expression levels. Apparently the 3' end of an RNA is a determinant of steady-state mRNA levels and, in turn, the subsequent production of the protein product.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.

The addition of glucose to the medium of Tetrahymena thermophila results in a 7-fold repression of galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase). The presence of millimolar amounts of the catecholamines dopa, dopamine, norepinephrine, and epinephrine or the hormone glucagon also results in the repression of galactokinase in the absence of glucose. The addition of millimolar amounts of adrenergic agonists (isoproterenol, tyramine, 2-amino-6,7-dihydroxytetrahydronaphthalene) results in significant repression of galactokinase in the absence of glucose; concentrations of 2-amino-6,7-dihydroxytetrahydronaphthalene less than or equal to 10(-4) M result in a derepression of galactokinase specific activity. Addition of adrenergic antagonists (propranolol, dichloroisoproterenol) have no effect on galactokinase activity at concentrations less than 10(-4) M but do arrest cell growth at greater concentrations. The addition of the cAMP analogs caffeine or theophylline in millimolar amounts results in repression of galactokinase activity; however, cell growth is greatly slowed or completely arrested at these concentrations. Analysis of the repression response of several mutants demonstrates that mutants deficient in catecholamine biosynthesis are altered in their regulation of galactokinase. Measurements of intracellular cAMP levels for 0-24 h following the addition of several of the above compounds to exponentially growing cells did not demonstrate any change over this period. Measurement of intracellular cAMP levels for 24 h following the addition of glucose or galactose to exponentially growing wild-type and mutant cell strains did not demonstrate any difference in cAMP concentrations over this period although a wide range of galactokinase activity was exhibited. Starvation of wild-type cells prior to the addition of glucose in minimal medium without added carbohydrate resulted in a significant increase in cAMP following the addition of glucose. This increase is demonstrated to be dependent upon the ability of the cells to resume division after the arrest of growth and is not correlated with galactokinase regulation. These results support the conclusion that cAMP is not involved in the repression of galactokinase gene expression initiated by glucose or hormone-like effectors and demonstrate the participation of an adrenergic control system in galactokinase regulation which is subordinate to the regulation by glucose. A possible model is discussed.

We have previously shown that the genes encoding the two subunits of Escherichia coli DNA gyrase are regulated in a manner which is dependent on DNA conformation. When the DNA encoding the gyrA and gyrB genes is relaxed, both genes are expressed at a high level; in negatively supercoiled DNA they are expressed at a low level. In this paper we describe fusions of both the gyrA and gyrB 5' sequences to the E. coli galactokinase gene. In such fusions we found that galactokinase can be induced by treating the cells with coumermycin A1, an inhibitor of DNA gyrase. Our results suggest that the regulation occurs at the transcriptional level and that only a small region of DNA is necessary for coumermycin-induced gene expression.

The rifampicin (Rif)-resistant RNA polymerase of phage T7 has proved invaluable for the exclusive over-expression, in Escherichia coli, of genes cloned downstream from the T7 phi 10 promoter [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82 (1985) 1074-1078]. Here, we demonstrate that the system can be extended to Gram-negative bacteria other than E. coli, by the use of compatible wide host range plasmids. As an example, the Rif-resistant in vivo synthesis and specific radiolabelling of E. coli galactokinase in Pseudomonas ATCC19151, is demonstrated. The incidental observation that 30 min after treatment with Rif, two polypeptides continue to be synthesized in plasmid-free Pseudomonas ATCC19151, indicates that these proteins are produced by very stable mRNA species.

Galactose utilization in Streptomyces lividans was shown to be controlled by an operon that is induced in the presence of galactose and repressed by glucose. Two promoters, galP1 and galP2, which direct transcription of two distinct polycistronic transcripts, have been identified. galP1 is located immediately upstream of the operon and is induced in the presence of galactose. This promoter directs transcription of the galT, galE, and galK genes. The second promoter, galP2, is located within the operon just upstream of the galE gene. This promoter is responsible for constitutive transcription of the galE and galK genes. Comparison of the S. lividans gal operon to the Escherichia coli gal operon indicates the presence of a constitutive promoter positioned upstream of galE in both operons. We suggest that coupling the operon's constitutive promoter to the galE gene fulfills a physiological requirement for constitutive UDPgalactose 4-epimerase expression in Streptomyces.

A mutant of Escherichia coli which is more resistant to shortwave UV light than its wild-type parent strain and which can synthesise DNA polymerase I constitutively has been further analysed. It carries two mutational alleles which are located about 1.5 min apart and cotransducible by P1 with the argH locus. The two mutational alleles have been segregated and their analysis shows that one of them is responsible for UV hyper-resistance whereas the other mutation confers UV sensitivity. Recombinant plasmids carrying various sections of the polA regulatory region, linked to a galK gene, were introduced into the mutant strains. Analysis of galactokinase shows that the enzyme activity in the UV hyper-resistant mutant is increased. The results suggest that the synthesis of DNA polymerase I in E. coli is inducible.

Seven dominant mutations showing greatly enhanced resistance to the glucose repression of galactokinase synthesis have been isolated from GAL81 mutants, which have the constitutive phenotype but are still strongly repressible by glucose for the synthesis of the Leloir enzymes. These glucose-resistant mutants were due to semidominant mutations at either of two loci, GAL82 and GAL83. Both loci are unlinked to the GAL81- gal4, gal80, or gal7 X gal10 X gal1 locus or to each other. The GAL83 locus was mapped on chromosome V at a site between arg9 and cho1. The GAL82 and GAL83 mutations produced partial resistance of galactokinase to glucose repression only when one or both of these mutations were combined with a GAL81 or a gal80 mutation. The GAL82 and GAL83 mutations are probably specific for expression of the Leloir pathway and related enzymes, because they do not affect the synthesis of alpha-D-glucosidase, invertase, or isocitrate lyase.

Mutants able to utilize 5'-AMP or cyclic AMP as the adenine source were isolated from an ade6 ade10 double mutant by ethyl methane sulfonate mutagenesis. A single amp1 mutation, primarily selected on 5'-AMP medium, confers the phenotype for utilization of exogenous 5'-AMP as the adenine source. From the ade6 ade10 amp1 triple mutant, a mutant able to utilize cyclic AMP was isolated, and the mutant phenotype was proven to be due to the simultaneous occurrence of triple mutations designated as cam1, cam2, and cam3. The cam3 mutation, but not cam1 or cam2, also confers the phenotype for utilizing 5'-AMP, the same phenotype as the amp1 mutation. All of these mutations are recessive to the respective wild-type counterparts. Cells having the ade6 ade10 amp1 cam1 cam2 cam3 genotype showed significant ability to take up exogenous cyclic AMP, whereas no differences were observed in cyclic AMP phosphodiesterase activity in comparison with that of the original strains used in the mutant isolation. Since glucose severely repressed galactokinase synthesis in the constitutive GAL81 mutant having the ade6 ade10 amp1 cam1 cam2 cam3 genotype, irrespective of the presence or absence of cyclic AMP in the medium, it was suggested that cyclic AMP is not involved in the mechanism of catabolite repression in Saccharomyces cerevisiae. It does, however, have a stimulative effect on the galactokinase synthesis in the GAL81 mutant in the absence of glucose.

In yeast cells galactosamine in concentrations of 0.1 1M partially inhibits the synthesis of RNA but has little effect on the protein synthesis. In vivo and in vitro studies show that galactosamine is metabolized in yeast to UDP-N-acetylhexosamines but at a reduced rate, compared to the metabolism of galactose. The addition of galactosamine to growing yeast cells leads to the induction of the galactose pathway enzymes. Studies using different mutants in the galactose genes provide evidence that galactosamine is an inducer of the galactose structural genes in yeast. The same degree of induction of galactokinase and galactotransferase, found when galactose or galactosamine were used as inducers, supports the model of coordinated regulation in the expression of the structural genes for the galactose pathway enzymes in yeast.

The integration host factor (IHF) binds to a site (ihf) that overlaps the -35 region of the phage lambda late rightward promoter (p'R). This interaction represses p'R-promoted transcription, both in vivo and in vitro. In vivo repression was observed when a plasmid carrying both p'R and the galK reporter gene was transfected into IHF+ or IHF- hosts. In vitro repression of transcription by IHF was observed only with linear, but not with supercoiled wild-type p'R templates. When binding to ihf, IHF imposes a strong bend on the DNA and protects this site from cleavage by neocarzinostatin, pancreatic DNase I, and hydroxyl radicals, as assessed by footprinting experiments. Both the functional and nonfunctional p'R mutants, in which the upstream part of the -35 region was replaced by an EcoRI linker, show modified behavior toward IHF. Some are more sensitive to IHF-mediated repression, even in the supercoiled form, while others have lost their affinity for IHF. We conclude that IHF binding depends not only on the consensus ihf sequence, but also on a suitable combination of the sequences of both ihf and neighboring regions, together with the DNA conformation, which includes both natural and imposed bends in DNA and the degree of supercoiling. Based on most of the present data, it is difficult to predict the relationship between the ihf sequence and IHF interaction, since two very different sequences (less than 50% homology) show strong IHF binding, whereas very similar sequences (80-87% homology) show a very different behavior. However, the hydroxylradical footprinting data show that three A + T-rich sequences are protected by IHF: the central sequence, which overlaps the -35 region of p'R, and two flanking sequences removed by one helix turn. All three sequences are located on the same face of the helix, and the amino acid side chains of IHF seem to occupy the narrow minor groove. A novel consensus sequence is proposed.

A total of 37 recessive mutations showing enhanced resistance to the glucose repression of galactokinase synthesis have been isolated by a selection procedure with a GAL81 gal7 double mutant. These mutations were grouped into three different complementation classes. One class, reg1, contains mutants arising from mutations at a site close to, but complementing, the gal3 locus. The reg1 mutant also showed resistance to the glucose repression of invertase synthesis but not to that of alpha-D-glucosidase. The two other classes were identified as arising from recessive mutations at the GAL82 locus and the GAL83 locus, respectively, at which various dominant mutations were isolated previously. When in a constitutive background due to the GAL81 or gal80 mutation, the GAL82 and GAL83 mutations did not show a mutually additive effect on the resistance to glucose repression of galactokinase synthesis, while the reg1 and GAL82 (or GAL83) mutations did. Based upon the specific behavior of cells with various genotypes for the above genes in response to the concentration of galactose and glucose in the medium, we propose a model involving three independent circuits for glucose signals in the regulation of the structural genes for the galactose pathway enzymes.

During the galactose adaptation period of a Saccharomyces cerevisiae strain bearing a naturally occurring gal3 allele, we found a longer induction lag and slower rate of accumulation of GAL10 and MEL1 RNAs compared to wild-type strains. A strain of genotype gal3 gal1 gal7 is noninducible for MEL1 gene expression, but this expression block is bypassed by overexpression of the GAL4 gene or by deletion of the GAL80 gene, either of which causes a constitutive phenotype. An otherwise wild-type strain that bears a chromosomal gal3 gene disruption mutation does not produce wild-type GAL3 RNA and exhibits induction comparable to a strain bearing the naturally occurring gal3. Based on this array of results, we conclude that the GAL3 gene product executes its function at a point before GAL4 mediated transcription of the GAL1-10-7 and MEL1 genes. Thus, the data are consistent with the previously advanced hypothesis that the GAL3 gene product functions to synthesize the inducer or coinducer molecule. In experiments in which the presence of either the plasmid-carried cloned GAL3 gene or the plasmid-carried cloned GAL1-10-7 genes allows MEL1 induction of a gal3 gal1 gal7 cell, we find that loss of the plasmid results in the shutoff of MEL1 expression even when galactose is continuously present. Either GAL3 function or GAL1-10-7 functions are therefore required for both the initiation and the maintenance of the induced state. Since the strains bearing either the naturally occurring gal3 allele or the gal3 disruption (null) allele do induce, the plasmid loss experiments indicate the existence of two completely independent induction initiation-maintenance pathways, one requiring GAL3 function, the other requiring GAL1-10-7 function. Finally, Northern blot analysis reveals two major GAL3 transcripts that differ in size by approximately 500 nucleotides.

A 'phase-shift' translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BclI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the lambda cro gene. The lambda cro gene is expressed from promoter pR and controlled by a thermosensitive (cI857) lambda repressor. The usefulness of the expression vector was demonstrated using a galK gene lacking the ATG start codon and fusing this to the pR promoter and ATG start codon of the lambda cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5'-GATC-3') at the N terminus (provided, for example, by a BamHI linker). The lambda cI857-pR-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful lambda pR promoter and the efficient lambda repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.

The presence of potential hairpin structures H1, H2, H3 in the leader region of a spinach rDNA operon led us to postulate that this operon is regulated by premature termination. The mechanism would be controlled by the presence or absence of ribosomes translating a leader peptide. In vitro synchronized transcription by E. coli RNA polymerase shows that pauses do occur in the leader region. By their sizes, the transient transcripts could correspond to pauses on H1 and H2 as predicted by the model in the absence of ribosomes. The complete leader sequence (pKOPH) and the leader sequence with the hairpin structures deleted (pKOP) have been used to the GalK gene in the pK01 plasmid. The resulting plasmids have been used to transform a GalK- E. coli strain. Measurements of GalK expression show that the promoter region of spinach chloroplast rDNA is neither subjected to the growth rate nor to the stringent control. However, under growth conditions leading to an excess of free ribosomes, the expression of GalK gene appears systematically to be reduced in pKOPH when compared with that of pKOP. These results are consistent with a role of the leader region in a translation-mediated attenuation of the chloroplast rDNA expression.

Using a plasmid containing a transcriptional fusion in which the E.coli galK gene is expressed from the trfB promoter of broad host range plasmid RK2 we show that transcription from the trfB promoter is repressed by the products of both the trfB and korB genes as we have previously predicted from the sequence homology of the trfA and trfB promoters and the fact that the trfA promoter is regulated by trfB and korB. These loci, trfB and korB are normally transcribed from the trfB promoter. Thus the trfB incC korB operon of RK2 is doubly autogeneously regulated. In addition, we describe the isolation and characterization of a mutant trfA promoter which has become insensitive to repression by trfB as a result of a point mutation within the inverted repeat sequence previously predicted to be the trfB protein binding site. These results provide strong evidence for our previously proposed model for control of transcription from the trfA and trfB promoters.

We have presented the results of our studies of the expression of the CYC genes from plasmids. All our data indicate that the levels of expression and the regulation of expression are very similar for the plasmid-borne genes and the chromosomal genes when care is taken to construct the appropriate plasmids. The usefulness of these plasmids has been demonstrated: mutations affecting regulatory sites adjacent to genes of interest have been constructed [such as the Xho I deletion and inversion in the YCpCYC1(2.4) plasmid] and selected [as in the case of the IS1 insertion into the YCpCYC7(2) plasmid], and these mutations have led us to some tentative conclusions about the location and nature of the regulatory sites of these genes. Furthermore, transformation with plasmids containing modified genes or fusions has permitted isolation of genomic regulatory mutants, as in the selection of lac+ suppressors of the lac- CYC1 1/x inversion carried on the YCpCYC1(2.4) 1/x plasmid. Although we cannot rule out the possibility that use of plasmids might cause us to miss a class of regulatory effects that can be propagated only along a chromosomal structure, we believe that the regulatory effects that we do observe can be more quickly and completely defined by working with plasmids. If any regulatory effects occur only on chromosomes, they can be studied more easily once the basic regulatory phenomena have been analyzed. The regulatory regions of the CYC1, CYC7, and TR2 genes that we have crudely mapped so far all exert their effects 100-300 bp away from the putative transcriptional starting sites. How the information in these regions is transmitted along the DNA is an intriguing question. We are engaged in a mutational analysis of these sites to locate them more precisely, to map second-site mutations that moderate the effects of the original mutations, to obtain genomic mutations that define the genes whose products interact with these sites, and to test combinations of genomic and plasmid mutations to define the sites with which regulatory elements interact. This approach should aid our understanding of the spatial relationships between yeast regulatory sites and transcriptional signals. Ultimately, obtaining mutations in regulatory genes, such as the mutations described here for the anaerobic regulation of TR2, will allow the cloning of these genes by complementation. This will lead to the isolation of the protein encoded and ultimately to an approach to the molecular mechanism of regulation through study of protein-DNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)