The anticancer agent 3-bromopyruvate: a simple but powerful molecule taken from the lab to the bedside.

At the beginning of the twenty-first century, 3-bromopyruvate (3BP), a simple alkylating chemical compound was presented to the scientific community as a potent anticancer agent, able to cause rapid toxicity to cancer cells without bystander effects on normal tissues. The altered metabolism of cancers, an essential hallmark for their progression, also became their Achilles heel by facilitating 3BP's selective entry and specific targeting. Treatment with 3BP has been administered in several cancer type models both in vitro and in vivo, either alone or in combination with other anticancer therapeutic approaches. These studies clearly demonstrate 3BP's broad action against multiple cancer types. Clinical trials using 3BP are needed to further support its anticancer efficacy against multiple cancer types thus making it available to more than 30 million patients living with cancer worldwide. This review discusses current knowledge about 3BP related to cancer and discusses also the possibility of its use in future clinical applications as it relates to safety and treatment issues.

Complete inventory of the yeast ABC proteins.

The complete sequence of the yeast genome predicts the existence of 29 proteins belonging to the ubiquitous ATP-binding cassette (ABC) superfamily. Using binary comparison, phylogenetic classification and detection of conserved amino acid residues, the yeast ABC proteins have been classified in a total of six clusters, including ten subclusters of distinct predicted topology and presumed distinct function. Study of the yeast ABC proteins provides insight into the physiological function and biochemical mechanisms of their human homologues, such as those involved in cystic fibrosis, adrenoleukodystrophy, Zellweger syndrome, multidrug resistance and the antiviral activity of interferons.

Life with 6000 genes.

The genome of the yeast Saccharomyces cerevisiae has been completely sequenced through a worldwide collaboration. The sequence of 12,068 kilobases defines 5885 potential protein-encoding genes, approximately 140 genes specifying ribosomal RNA, 40 genes for small nuclear RNA molecules, and 275 transfer RNA genes. In addition, the complete sequence provides information about the higher order organization of yeast's 16 chromosomes and allows some insight into their evolutionary history. The genome shows a considerable amount of apparent genetic redundancy, and one of the major problems to be tackled during the next stage of the yeast genome project is to elucidate the biological functions of all of these genes.

The composite genome of the legume symbiont Sinorhizobium meliloti.

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

In-frame recombination between the yeast H(+)-ATPase isogenes PMA1 and PMA2: insights into the mechanism of recombination initiated by a double-strand break.

Chimeric PMA1::PMA2 sequences, placed under the control of the PMA1 promoter, were constructed by in vivo recombination between a gapped linearized plasmid containing the PMA2 gene and four different fragments of the PMA1 gene. Correct in-frame assembly of the PMA sequences was screened by the expression of the lacZ reporter gene fused to the PMA2 coding region. Restriction and sequencing analysis of 35 chimeras showed that in all cases, the hybrid sequences was obtained as fusions between continuous sequences specific to PMA1 and PMA2, separated by a region of identity. In all but three cases, the junction sequences were not located at regions of greatest identity. Strikingly, depending on the PMA1 fragment used, junction distribution fell into two categories. In the first, the junctions were scattered over several hundreds of nucleotides upstream of the extremity of the PMA1 fragment, while in the second, they were concentrated at this extremity. Analysis of the alignment of the PMA1 and PMA2 sequences suggests that the distribution is not related to the size of the region of identity at the PMA1-PMA2 boundary but depends on the degree of identity of the PMA genes upstream of the region of identity, the accumulation of successive mismatches leading to a clustered distribution of the junctions. Moreover, the introduction of seven closely spaced mismatches near the end of a PMA1 segment with an otherwise-high level of identity with PMA2 led to a significantly increased concentration of the junctions near this end. These data show that a low level of identity in the vicinity of the common boundary stretch is a strong barrier to recombination. In contrast, consecutive mismatches or regions of overall moderate identity which are located several hundreds of nucleotides upstream from the PMA1 end do not necessarily block recombination.

Heavy metal transporters in Hemiascomycete yeasts.

We have compiled all known heavy metal transporters of the yeast Saccharomyces cerevisiae and identified their orthologs in four other species spanning the entire Hemiascomycete phylum. The 213 transporters belong to 27 distinct phylogenetic families distributed within the three classes: channels, secondary porters (permeases) and transport ATPases. They are present in all cellular membranes: plasma membranes, vacuoles, mitochondria, endoplasmic reticulum, nucleus, Golgi and various cytoplasmic vesicles. The major physiological heavy metals transported are: iron, manganese, zinc, copper, arsenite and cadmium. The major subfamilies that comprise the highest number of transporters are Siderophore-Iron Transporters (SIT) and CT2 (conjugated ABC transporters). They transport heavy metals (iron or cadmium, respectively) conjugated to organic chelators such as siderophores or glutathione. Both subfamilies are considerably amplified in the yeast Yarrowia lipolytica. The pattern of expansion and restriction of the subfamilies during the evolution of the different species is highly variable. The phylogenetic trees of the major transporters subfamilies distinguish homogenous clusters of transporters suggesting that possible different physiological or mechanistic functions evolved independently. We also validated the use of the Hemiascomycetes heavy metal transporters for identification of orthologs transporters in the pathogenic Basidiomycetes Cryptococcus neoformans.

Role of the yeast ABC transporter Yor1p in cadmium detoxification.

Growth of yeast strains, either deleted for the vacuolar ABC transporter Ycf1 or deleted for the plasma membrane ABC transporter Yor1p or overexpressing Yor1p, were compared for their sensitivity to cadmium. On solid medium cell death (or growth inhibition) was observed at cadmium concentrations higher than 100 microM when yeasts were grown at 30 degrees C for 24 h. However, for all tested strains cell death (or growth inhibition) was already observed at 40 microM cadmium when incubated at 23 degrees C for 60 h. Thus cadmium is more toxic to yeast at 23 degrees C than at 30 degrees C. At 23 degrees C, the Deltayor1 strain grew more slowly than the wild-type strain and the double Deltayor1, Deltaycf1 deleted strain was much more sensitive to cadmium than each single Deltayor1 or Deltaycf1 deletant. Overexpression of Yor1p in a Deltaycf1 strain restores full growth. Cadmium uptake measurements show that Deltaycf1 yeast strains expressing or overexpressing Yor1p store less cadmium than the corresponding Deltaycf1, Deltayor1 strain. The strains expressing Yor1p display an energy-dependent efflux of cadmium estimated for the yeast overexpressing Yor1p to be about 0.02 nmol 109Cd/mg protein/min. Yeast cells loaded with radiolabeled glutathione and then with radioactive cadmium displayed a twice-higher efflux of glutathione than that of cadmium suggesting that Yor1p transports both compounds as a bis-glutathionato-cadmium complex. All together, these results suggest that in addition to being accumulated in the yeast vacuole by Ycf1p, cadmium is also effluxed out of the cell by Yor1p.

Classification of all putative permeases and other membrane plurispanners of the major facilitator superfamily encoded by the complete genome of Saccharomyces cerevisiae.

On the basis of the complete genome sequence of the budding yeast Saccharomyces cerevisiae, a computer-aided analysis was carried out of all members of the major facilitator superfamily (MFS), which typically consists of permeases with 12 transmembrane spans. Analysis of all 5885 predicted open reading frames identified 186 potential MFS proteins. Binary sequence comparison made it possible to cluster 149 of them into 23 families. Putative permease functions could be assigned to 12 families, the largest including sugar, amino acid, and multidrug transport. Phylogenetic clustering of proteins allowed us to predict a possible permease function for a total of 119 proteins. Multiple sequence alignments were made for all families, and evolutionary trees were constructed for families with at least four members. The latter resulted in the identification of 21 subclusters with presumably tightly related permease function. No functional clues were predicted for a total of 41 clustered or unclustered proteins.

Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor.

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown "petite-negative" yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40 degrees C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles. 2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerol-grown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae. 3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the "petite-positive" yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126. 4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 muM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 muM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor. 5. We conclude that "petite-positive" and "petite-negative" yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.

Energy-dependent uptake of calcium by the yeast Schizosaccharomyces pombe.

1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.

Electrogenic proton ejection coupled to electron transport through the energy-conserving site 2 and K+/H+ exchange in yeast mitochondria.

The proton ejection coupled to electron flow from succinate and/or endogenous substrate(s) to cytochrome c using the impermeable electron acceptor ferricyanide is studied in tightly coupled mitochondria isolated from two strains of the yeast Saccharomyces cerevisiae. (1) The observed H+ ejection/2e- ratio approaches an average value of 3 when K+ (in the presence of valinomycin) is used as charge-compensating cation. (2) In the presence of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone, an H+ ejection/2e- ratio of 2 is observed. (3) The low stoichiometry of 3H+ ejected (instead of 4) per 2e- and the high rate of H+ back-decay (0.1615 ln delta (ngatom)H+/s and a half-time of 4.6 s for 10 mg protein) into the mitochondrial matrix are related to the presence of an electroneutral K+/H+ antiporter which is demonstrated by passive swelling experiments in isotonic potassium acetate medium.

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles.

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.

Regulation of the expression of the H(+)-ATPase genes PMA1 and PMA2 during growth and effects of octanoic acid in Saccharomyces cerevisiae.

A peak of plasma membrane H(+)-ATPase activity during exponential growth is correlated with the expression of the PMA1 gene as monitored by measurements of the beta-galactosidase activity from a PMA1-lacZ fusion. This peak of activity is also correlated to the content of the H(+)-ATPase protein in yeast plasma membrane as shown by quantitative immunodetection. The PMA2-lacZ fusion assay indicates that the expression of the PMA2 gene is activated somewhat later during exponential phase but under all circumstances its activity remains at least 500-fold lower than that of the PMA1-lacZ fusion. A slight but significant stimulation of ATPase activity by low concentrations of octanoic acid coincides with a decrease in the PMA1 gene expression. It is concluded that octanoic acid stimulates de PMA1 ATPase activity by posttranslational mechanisms.

Active efflux by multidrug transporters as one of the strategies to evade chemotherapy and novel practical implications of yeast pleiotropic drug resistance.

Mankind is faced by the increasing emergence of resistant pathogens, including cancer cells. An overview of the different strategies adopted by a variety of cells to evade chemotherapy is presented, with a focus on the mechanisms of multidrug transport. In particular, we analyze the yeast network for pleiotropic drug resistance and assess the potentiality of this system for further understanding of the mechanism of broad specificity and for development of novel practical applications.

Sequencing the yeast genome: the European effort.

For ethical, practical and economic reasons, scientists have traditionally relied on model organisms for biological research. Although model organisms do not always quite constitute the 'real thing', the significant advantages of their use contribute to making their study a viable alternative. The decision to use a specific model, particularly in large-scale studies such as genome projects, will be governed not only by biological consideration, but also by the prevailing financial and organizational infrastructure and expertise of the research community.

The RNA polymerase I initiation site and the external transcribed spacer of the fission yeast Schizosaccharomyces pombe ribosomal RNA genes.

A 5.45-kb fragment containing the 5' end of the ribosomal RNA transcriptional unit from the fission yeast Schizosaccharomyces pombe was cloned in the yeast-E. coli shuttle vector YEp13. The transcription start point was mapped by R looping and S1 nuclease protection. The sequence of the entire external transcribed spacer (ETS) and its flanking regions was determined. Comparison of the sequence around the transcription start point with those of four budding yeasts (Saccharomycetoideae) reveals a consensus sequence from position -9 to -4 from the start. This sequence is likely to be an important element of the promoter for yeast RNA polymerase I (Pol.I). Comparison of all known Pol.I promoter sequences reveals a strong bias for nucleotides (nt) at several positions between -16 and +10. These nt may have a critical role in the transcription initiation process. The S. pombe ETS, which comprises 1355 bp, is significantly longer than those of the budding yeasts and lacks any significant sequence homology with the Saccharomyces cerevisiae ETS. R-loop analysis reveals a putative processing site within the ETS of S. pombe.

The suppressor gene scl1+ of Saccharomyces cerevisiae is essential for growth.

In Saccharomyces cerevisiae, the SCL-1 mutation is a dominant suppressor of the cycloheximide-resistant, temperature-sensitive (ts) lethal mutation, crl3 [McCusker and Haber, Genetics 119 (1988a) 303-315]. The wild-type scl1+ gene was isolated by screening subclones of the 35-kb region between TRP5 and LEU1 for restoration of the ts phenotype in an SCL1-1 crl3-2 strain. The scl1+ mRNA is about 900 nt long and encodes an open reading frame of 810 bp. The polypeptide deduced from scl1+ possesses a putative secretory signal peptide. The 5'-noncoding region may be under multiple controls, since it contains significant homology to the consensus sequences for the DNA-binding proteins, GCN4, GFI and, possibly, TUF. Gene disruption of scl1+ demonstrates that it is an essential gene.

Novel target genes of the yeast regulator Pdr1p: a contribution of the TPO1 gene in resistance to quinidine and other drugs.

The yeast transcription factor Pdr1p regulates the expression of a number of genes, several of which encode ATP-driven transport proteins involved in multiple drug resistance. Among 20 genes containing binding consensus sequences for the transcription factor Pdr1p in their promoter, we studied more particularly the regulation and function of PDR16 (involved in phospholipid synthesis), TPO1 (involved in vacuolar transport of polyamines), YAL061W (homologous to polyol dehydrogenases) and YLR346C (unknown function). We found that the regulation of these four genes depends on Pdr1p, since promoter activities studied by lacZ fusion analysis and mRNA levels studied by Northern blotting analysis changed upon deletion or hyperactivation by the pdr1-3 mutant of this transcription factor. The drug sensitivity of the strains deleted for these genes revealed that TPO1, a gene previously found to be involved in spermidine resistance and vacuolar polyamine transport, is a determinant of multidrug transporter since it also mediates growth resistance to cycloheximide and quinidine. This resistance pattern overlapped with that of YOR273C, a homolog of TPO1. These two homologous transporters are thus bona fide members of the phylogenetic subfamily DHA1 (drug/proton antiport TC 2.A.1. 2) of the major facilitator superfamily. Both YOR273C and TPO1 as well as at least one other determinant involved in the yeast pleiotropic drug resistance network contribute to resistance to a quinoline-containing antimalarial drug.

Analysis of second-site mutations that suppress the multiple drug resistance phenotype of the yeast PDR1-7 allele.

The yeast PDR1 locus encodes a member of the C6 zinc cluster family of transcriptional regulatory proteins. Among the targets of PDR1 is the yeast PDR5 locus. The product of this gene is a member of the ATP-binding cassette (ABC) transmembrane protein family and plays a major role in inhibitor efflux. Mutations in PDR1 affect the relative level of PDR5 transcript and can therefore result in increased or decreased drug resistance. We isolated three second-site suppressors of a PDR1-7 semidominant hyper-resistant mutation. These mutants were drug hypersensitive, as compared with isogenic controls. Two of the three mutations contained alterations in a putative DNA-binding domain. Significantly, the mutant proteins exhibited reduced DNA-binding capacity.

Four years of post-genomic life with 6,000 yeast genes.

Four years after disclosure of the full yeast genome sequence, a series of resources including tens of thousands of mutant strains, plasmids bearing isolated genes and disruption cassettes are becoming publicly available. Deletions of each of the 6,000 putative yeast genes are being screened systematically for dozens of phenotypic traits. In addition, new global approaches such as DNA hybridization arrays, quantitative proteomics and two-hybrid interactions are being steadily improved. They progressively build up an immense computation network of billions of data points which will, within the next decade, characterize all molecular interactions occurring in a simple eukaryotic cell. In this process of acquisition of new basic knowledge, an international community of over 1,000 laboratories cooperates with a remarkable willingness to share projects and results.