Yeast cDNAs Research Articles

Functional characterization of polyol/monosaccharide transporter 1 in Lotus japonicus

Polyol/Monosaccharide Transporters (PLTs/PMTs) localized in the plasma membrane have previously been identified in plants. The physiological role and the functional properties of these proteins in legume plants are, however, unclear. Here we describe the functional analysis of LjPLT1, a plasma membrane-localized PLT protein from Lotus japonicus. The LjPLT1 gene was strongly expressed in the vascular tissue of roots, stems and leaves. Expression of the LjPLT1 cDNAs in yeast revealed that the protein functions as a broad-spectrum H+ -symporter for both linear polyols of sorbitol and mannitol, and cyclic polyol myo-inositol. It also catalyzes the transport of different hexoses, including fructose, glucose, galactose and mannose. Overexpression of LjPLT1 (OELjPLT1) results in inhibition of plant growth and a decrease in nodule nitrogenase activity in L. japonicus. The soluble sugars were increased in newly expanded leaves, roots and nodules but decreased in mature leaves in OELjPLT1 plants. In addition, the OELjPLT1 seedlings displayed an increased sensitivity to high content mannitol and boron toxicity, but neither drought nor salinity stresses. Taken together, the present study indicates that the LjPLT1 protein may participate in the translocation of hexoses/polyols to regulate multiple physiological and growth processes in L. japonicus.

Expression of Chlamydomonas reinhardtii CrGPDH2 and CrGPDH3 cDNAs in yeast reveals that they encode functional glycerol-3-phosphate dehydrogenases involved in glycerol production and osmotic stress tolerance

Glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P) and plays a central role in the synthesis of glycerol and triacylglycerides (TAGs). Osmotic stress has been shown to induce the accumulation of glycerol and TAGs in the green microalga Chlamydomonas reinhardtii. In a previous study, we identified three GPDH homologs (CrGPDH1, CrGPDH2, and CrGPDH3) in this microalga. We found that CrGPDH2 and CrGPDH3 were expressed in response to 200 mM NaCl treatment, suggesting that these two genes play roles in glycerol and TAGs synthesis and in osmotic stress tolerance. In this study, we report on the functional characterization of CrGPDH2 and CrGPDH3. A concentration of NaCl as low as 5 mM for 5 min was sufficient to induce the expressions of both genes. We mapped the cDNA ends of CrGPDH2 and CrGPDH3 using RLM-RACE and cloned their full-length cDNAs. The expression of these two cDNAs in the Saccharomyces cerevisiae gpd1Δgpd2Δ double mutant confirmed that both CrGPDH2 and CrGPDH3 have GPDH activity. The genetic complementation analysis revealed that CrGPDH2 and CrGPDH3 were able to restore glycerol production and rescue the salt sensitivity of this mutant. Compared with CrGPDH3, CrGPDH2 conferred higher glycerol production and greater salt tolerance when expressed in the gpd1Δgpd2Δ double mutant. Together, these findings show that CrGPDH2 and CrGPDH3 encode functional homologs of the S. cerevisiae GPD1 gene that is involved in glycerol synthesis and osmotic stress tolerance.

Molecular cloning and characterization of (+)-epi-α-bisabolol synthase, catalyzing the first step in the biosynthesis of the natural sweetener, hernandulcin, in Lippia dulcis

Hernandulcin, a C15 sesquiterpene ketone, is a natural sweetener isolated from the leaves of Lippia dulcis. It is a promising sugar substitute due to its safety and low caloric potential. However, the biosynthesis of hernandulcin in L. dulcis remains unknown. The first biochemical step of hernandulcin is the synthesis of (+)-epi-α-bisabolol from farnesyl diphosphate, which is presumed to be catalyzed by a unique sesquiterpene synthase in L. dulcis. In order to decipher hernandulcin biosynthesis, deep transcript sequencings (454 and Illumina) were performed, which facilitated the molecular cloning of five new sesquiterpene synthase cDNAs from L. dulcis. In vivo activity evaluation of these cDNAs in yeast identified them as the sesquiterpene synthases for α-copaene/δ-cadinene, bicyclogermacrene, β-caryophyllene, trans-α-bergamotene, and α-bisabolol. The engineered yeast could synthesize a significant amount (∼0.3mg per mL) of α-bisabolol in shake-flask cultivation. This efficient in vivo production was congruent with the competent kinetic properties of recombinant α-bisabolol synthase (Km 4.8μM and kcat 0.04s−1). Detailed chemical analyses of the biosynthesized α-bisabolol confirmed its configuration to be (+)-epi-α-bisabolol, the core skeleton of hernandulcin. These results demonstrated that enzymatic, stereoselective synthesis of (+)-epi-α-bisabolol can be achieved, promising the heterologous production of a natural sweetener, hernandulcin.

Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTERS 1 and 2: fructose and xylitol/H+ symporters in pollen and young xylem cells

The genome of Arabidopsis thaliana contains six genes, AtPMT1 to AtPMT6 (Arabidopsis thaliana POLYOL/MONOSACCHARIDE TRANSPORTER 1–6), which form a distinct subfamily within the large family of more than 50 monosaccharide transporter-like (MST-like) genes. So far, only AtPMT5 [formerly named AtPLT5 (At3g18830)] has been characterized and was shown to be a plasma membrane-localized H+-symporter with broad substrate specificity. The characterization of AtPMT1 (At2g16120) and AtPMT2 (At2g16130), two other, almost identical, members of this transporter subfamily, are presented here. Expression of the AtPMT1 and AtPMT2 cDNAs in baker's yeast (Saccharomyces cerevisiae) revealed that these proteins catalyse the energy-dependent, high-capacity transport of fructose and xylitol, and the transport of several other compounds with lower rates. Expression of their cRNAs in Xenopus laevis oocytes showed that both proteins are voltage-dependent and catalyse the symport of their substrates with protons. Fusions of AtPMT1 or AtPMT2 with the green fluorescent protein (GFP) localized to Arabidopsis plasma membranes. Analyses of reporter genes performed with AtPMT1 or AtPMT2 promoter sequences showed expression in mature (AtPMT2) or germinating (AtPMT1) pollen grains, as well as in growing pollen tubes, hydathodes, and young xylem cells (both genes). The expression was confirmed with an anti-AtPMT1/AtPMT2 antiserum (αAtPMT1/2) raised against peptides conserved in AtPMT1 and AtPMT2. The physiological roles of the proteins are discussed and related to plant cell wall modifications.

The role of novel and known telomeric chromatin‐associated proteins in suppression of DNA instability in Ku mutants of S. cerevisiae

The Yku70/Yku80 protein heterodimer is essential for precise rejoining of the ends of broken chromosomes after cells are exposed to DNA damaging agents such as X-rays or strand-breaking chemicals. The complex is also a component of the specialized protein structures normally found at chromosome ends, or telomeres, and is believed to serve a protective function there. Yeast cells (Saccharomyces cerevisiae) with either the YKU70 or YKU80 gene inactivated are viable, but have unstable chromosome ends. This instability is exacerbated at slightly elevated growth temperatures and cells arrest growth in G2 phase of the cell cycle and ultimately lose viability. Interestingly, high temperature killing of yku70 mutants is prevented when intracellular levels of specific subunits of the telomerase DNA replication complex are increased. For example, overexpression of TLC1, the RNA subunit of telomerase, or EST2, which encodes the polymerase subunit, have both been found to prevent death of yku70 and yku80 mutants. The mechanism of this rescue is unknown. My research project involves an analysis of the genes and mechanism(s) involved using two approaches. The project will begin by investigating the role of telomeric chromatin proteins Sir3, Sir4, Rif1, and Rif2 in yku70 mutant rescue. The expression of these proteins will be elevated and the rescue effect at high temperature measured. Next, novel genes involved in Ku mutant rescue at high temperature will be explored using a library screen. A gene library, which uses a regulatory GAL1 promoter to express yeast cDNAs at high levels will be used. These two approaches will lead to a better understanding of the mechanism of telomere stabilization by telomeric chromatin proteins and the identification of new genes involved in telomere stability.

Molecular cloning and characterization of genes encoding two microsomal oleate desaturases ( FAD2) from olive

Two different cDNA sequences, designated OepFAD2-1 and OepFAD2-2, encoding two microsomal oleate desaturases (FAD2) have been isolated from olive ( Olea europaea cv. Picual) using a PCR approach. Both deduced amino acid sequences showed the three histidine boxes characteristic of all membrane-bound desaturases, and possess a C-terminal endoplasmic reticulum retention signal. Phylogenetic analysis shows that OepFAD2-1 and OepFAD2-2 are grouped with other plant FAD2 sequences. Functional expression of the corresponding FAD2 cDNAs in yeast confirmed that they encode microsomal oleate desaturases. Genomic Southern blot analysis is consistent with the presence of at least two copies of each OepFAD2 gene in the olive genome. OepFAD2-1 transcript was strongly detected in very young seeds and in leaves, showing low levels in mesocarps, while the transcript of the OepFAD2-2 gene was moderately expressed in developing seeds, ripening mesocarp and leaves. These expression data suggest differential functions for the two olive microsomal oleate desaturase genes, with FAD2-1 possibly responsible for the desaturation of reserve lipids in the young seed, while FAD2-2 may be mainly involved in storage lipid desaturation in the mature seeds and the mesocarp.

Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs

A yeast cDNA expression library was screened to identify genes and cellular processes that influence fungal sensitivity to a plant antimicrobial peptide. A plasmid-based, GAL1 promoter-driven yeast cDNA expression library was introduced into a yeast genotype susceptible to the antimicrobial peptide MiAMP1 purified from Macadamia integrifolia. Following a screen of 20,000 cDNAs, three yeast cDNAs were identified that reproducibly provided transformants with galactose-dependent resistance to MiAMP1. These cDNAs encoded a protein of unknown function, a component (VMA11) of the vacuolar H(+)-ATPase and a component (cytochrome c oxidase subunit VIa) of the mitochondrial electron transport chain, respectively. To identify genes that increased sensitivity to MiAMP1, the yeast cDNA expression library was introduced into a yeast mutant with increased resistance to MiAMP1. From 11,000 cDNAs screened, two cDNA clones corresponding to a ser/thr kinase and a ser/thr phosphatase reproducibly increased MiAMP1 susceptibility in the mutant in a galactose-dependent manner. Deletion mutants were available for three of the five genes identified but showed no change in their sensitivity to MiAMP1, indicating that these genes could not be detected by screening of yeast deletion mutant libraries. Yeast cDNA expression library screening therefore provides an alternative approach to gene deletion libraries to identify genes that can influence the sensitivity of fungi to plant antimicrobial peptides.

Using yeast to place human genes in functional categories

The availability of the draft sequence of the human genome has created a pressing need to assign functions to each of the 35,000 or so genes that it defines. One useful approach for this purpose is to use model organisms for both bioinformatic and functional comparisons. We have developed a complementation system, based on the model eukaryote Saccharomyces cerevisiae, to clone human cDNAs that can functionally complement yeast essential genes. The system employs two regulatable promoters. One promoter, tetO (determining doxycycline-repressible expression), is used to control essential S. cerevisiae genes. The other, p MET3 (which is switched off in the presence of methionine), is employed to regulate the expression of mammalian cDNAs in yeast. We have demonstrated that this system is effective for both individual cDNA clones and for cDNA libraries, permitting the direct selection of functionally complementing clones. Three human cDNA libraries have been constructed and screened for clones that can complement specific essential yeast genes whose expression is switched off by the addition of doxycycline to the culture medium. The validity of each complementation was checked by showing that the yeast cells stop their growth in the presence of doxycycline and methionine, which represses the expression of the yeast and mammalian coding sequence, respectively. Using this system, we have screened 25 tetO replacement strains and succeeded in isolating human cDNAs complementing six essential yeast genes. In this way, we have uncovered a novel human ubiquitin-conjugating enzyme, have isolated a human cDNA clone that may function as a signal peptidase and have demonstrated that the functional segment of the human Psmd12 proteosome sub-unit contains a PINT domain.

Molecular characterization of a heteromeric ATP-citrate lyase that generates cytosolic acetyl-coenzyme A in Arabidopsis.

Acetyl-coenzyme A (CoA) is used in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals including waxes, isoprenoids, stilbenes, and flavonoids. The source of cytosolic acetyl-CoA is unclear. We identified two Arabidopsis cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase (ACL). Coexpression of these cDNAs in yeast (Saccharomyces cerevisiae) confers ACL activity, indicating that both the Arabidopsis genes are required for ACL activity. Arabidopsis ACL is a heteromeric enzyme composed of two distinct subunits, ACLA (45 kD) and ACLB (65 kD). The holoprotein has a molecular mass of 500 kD, which corresponds to a heterooctomer with an A(4)B(4) configuration. ACL activity and the ACLA and ACLB polypeptides are located in the cytosol, consistent with the lack of targeting peptides in the ACLA and ACLB sequences. In the Arabidopsis genome, three genes encode for the ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; and ACLA-3, At1g09430), and two genes encode the ACLB subunit (ACLB-1, At3g06650 and ACLB-2, At5g49460). The ACLA and ACLB mRNAs accumulate in coordinated spatial and temporal patterns during plant development. This complex accumulation pattern is consistent with the predicted physiological needs for cytosolic acetyl-CoA, and is closely coordinated with the accumulation pattern of cytosolic acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate. Taken together, these results indicate that ACL, encoded by the ACLA and ACLB genes of Arabidopsis, generates cytosolic acetyl-CoA. The heteromeric organization of this enzyme is common to green plants (including Chlorophyceae, Marchantimorpha, Bryopsida, Pinaceae, monocotyledons, and eudicots), species of fungi, Glaucophytes, Chlamydomonas, and prokaryotes. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that a evolutionary fusion of the ACLA and ACLB genes probably occurred early in the evolutionary history of this kingdom.

A micro-scale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae

Methods have been developed aimed at applying at high-throughput technology for expression of cloned cDNAs in yeast. Yeast is a eukaryotic host, which produces soluble recombinant proteins and is capable of introducing post-translational modifications of protein. It is, thus, an appropriate expression system both for the routine expression of various cDNAs or protein domains and for the expression of proteins, which are not correctly expressed in Escherichia coli. Here, we describe a standard system in Saccharomyces cerevisiae, based on a vector for intracellular protein expression, where the gene products are fused to specific peptide sequences (tags). These epitope tags, the N-terminal His 6 tag and the C-terminal StrepII tag, allow subsequent immunological identification and purification of the gene products by a two-step affinity chromatography. This method of dual-tagged recombinant protein purification eliminates contamination by degraded protein products. A miniaturization of the procedures for cloning, expression, and detection was performed to allow all steps to be carried out in 96-well microtiter plates. The system is, thus, suitable for automation. We were able to analyze the simultaneous protein expression of a large number of cDNA clones due to the highly parallel approach of protein production and purification. The microtiter plate technology format was extended to quantitative analysis. An ELISA-based assay was developed that detects StrepII-tagged proteins. The application of this high-throughput expression system for protein production will be a useful tool for functional and structural analyses of novel genes, identified by the Human Genome Project and other large-scale sequencing projects.

Molecular cloning and expression in yeast of 2,3-oxidosqualene-triterpenoid cyclases from Arabidopsis thaliana.

A vast array of triterpenes are found in living organisms in addition to lanosterol and cycloartenol, which are involved in sterol biosynthesis in non-photosynthetic and photosynthetic eukaryotes respectively. The chemical structure of these triterpenes is determined by a single step catalysed by 2,3-oxidosqualene-triterpene cyclases. The present study describes cloning and functional expression in yeast of several OS-triterpene cyclases. Three Arabidopsis thaliana cDNAs encoding proteins (ATLUP1, ATLUP2, ATPEN1) 57%, 58% and 49% identical to cycloartenol synthase from the same plant were isolated. Expression of these cDNAs in yeast showed that the recombinant proteins catalyse the synthesis of various pentacyclic triterpenes. Whereas ATLUP1 is essentially involved in the synthesis of lupeol, ATLUP2 catalyses the production of lupeol, beta- and alpha-amyrin (in a 15:55:30 ratio). ATLUP2 is therefore a typical multifunctional enzyme. Under the same conditions, ATPEN1 did not lead to any product. Systematic sequencing of the Arabidopsis genome has led to genomic sequences encoding proteins identical to the above triterpene synthases. ATLUPI and ATLUP2 are representative of a small subfamily (A) of at least five genes, whereas ATPEN1 is representative of a subfamily (B) of at least seven genes. The number of introns is characteristic of each subfamily. Whereas genes of family A possess 17 exons and 16 introns, genes of the subfamily B contain 14 exons and 13 introns. The size of each exon is remarkably conserved within each subfamily whereas that of each intron appears to be highly variable. Organization of the genes, sequences and functions of the deduced proteins are discussed in evolutionary terms.

Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal-type and high-oleic varieties of sunflower (Helianthus annuus L.)

In addition to the normal-type sunflower (Helianthus annuus L.) where linoleic acid is the major seed fatty acid, a dominant negative high-oleic mutant with oleic acid as the predominant fatty acid was previously obtained. We report the isolation and characterization of three different cDNA sequences, designated Ha89FAD2-1, Ha89FAD2-2, and Ha89FAD2-3, encoding sunflower microsomal oleate desaturases (FAD2), using a PCR strategy. All three deduced amino acid sequences showed significant homology to the known plant FAD2 sequences. Genomic Southern blot analysis revealed that at least one copy of each of these genes is present in the sunflower genome, except for the FAD2-1 gene from the high-oleic mutant, which might be duplicated. The FAD2-2 and FAD2-3 genes were weakly expressed in all tissues studied from both varieties. In contrast, the FAD2-1 gene was expressed strongly and exclusively in developing embryos of normal-type sunflower, whereas its expression in high-oleic developing embryos was drastically reduced. Functional expression of the corresponding cDNAs in yeast confirmed that they encode microsomal oleate desaturases. Furthermore, the FAD2-1 gene from the high-oleic variety also expresses a fully active enzyme. These results suggest that the high-oleic mutation in sunflower interferes with the regulation of the transcription of the seed-specific FAD2 gene.

Increasing Expression of P450 and P450-Reductase Proteins from Monocots in Heterologous Systems

Monocotyledonous crop plants are usually more resistant to herbicides than grass weeds and most dicots. Their resistance to herbicides is mediated in many cases by P450 oxygenases. Monocots thus constitute an appealing source of P450 enzymes for manipulating herbicide resistance and recombinant forms of the major xenobiotic metabolizing mooxygenases are potential tools for the optimization of new active molecules. We report here the isolation and functional characterization of the first P450 and P450 reductase coding sequences from wheat. The first attempts at expressing these cDNAs in yeast and tobacco led to levels of protein, which were extremely low, often not even detectable. The wheat P450 cDNAs were efficiently transcribed, but no protein or activity was found. Wheat coding sequences, like those of other monocots, are characterized by a high GC content and by a related strong bias of codon usage, different from that observed in yeast or dicots. Complete recoding of genes being costly, the reengineering their 5′-end using a single PCR megaprimer designed to comply with codon usage of the host was attempted. It was sufficient to relieve translation inhibition and to obtain good levels of protein expression. The same strategy also resulted in a dramatic increase in protein expression in tobacco. A basis for the success of such a partial recoding strategy, much easier and cheaper than complete recoding of the cDNA, is proposed.

Functional expression of two Arabidopsis aldehyde oxidases in the yeast Pichia pastoris.

To investigate the biochemical and enzymatic properties of two aldehyde oxidase (AO) isoforms of Arabidopsis thaliana, we expressed AAO1 and AAO2 cDNAs in a heterologous yeast (Pichia pastoris) system and successfully obtained the proteins in active forms. The expressed AAO1 and AAO2 proteins gave activity bands with the same mobilities on native gel electrophoresis and exhibited the same substrate preferences on zymograms with 8 aldehydes as those of AOalpha and AOgamma in Arabidopsis seedlings, respectively. Furthermore, anti-AAO1 and anti-AAO2 antibodies, which specifically recognize the seedling AOalpha and AOgamma, respectively, reacted with the AAO1 and AAO2 proteins produced in P. pastoris, respectively. These results indicate that these AO proteins are accurately produced in the yeast system, as in Arabidopsis seedlings. Using AO preparations from P. pastoris, the enzymatic properties of Arabidopsis AOalpha and AOgamma were investigated. AOalpha showed a relatively wide substrate specificity for 7 aldehydes tested, with high affinity to benzaldehyde and indole-3-aldehyde, while AOgamma could most efficiently oxidize naphthaldehyde. AOalpha was strongly inhibited by iodoacetate and KCN, while AOgamma was inhibited not only by iodoacetate and KCN but also by 2-mercaptethanol, dithiothreitol, menadion, and estradiol. AOalpha and AOgamma showed the highest activity at around 65 and 50 degrees C, respectively, and exhibited pH dependence around pH 8.0. These results indicate that the two AO isoforms in Arabidopsis seedlings have different enzymatic properties and may have different physiological roles in vivo.

Characterization of Human, Schizosaccharomyces pombe, and Candida albicans mRNA Cap Methyltransferases and Complete Replacement of the Yeast Capping Apparatus by Mammalian Enzymes

Human and fission yeast cDNAs encoding mRNA (guanine-N7) methyltransferase were identified based on similarity of the human (Hcm1p; 476 amino acids) and Schizosaccharomyces pombe (Pcm1p; 389 amino acids) polypeptides to the cap methyltransferase of Saccharomyces cerevisiae (Abd1p). Expression of PCM1 or HCM1 in S. cerevisiae complemented the lethal phenotype resulting from deletion of the ABD1 gene, as did expression of the NH2-terminal deletion mutants PCM1(94-389) and HCM1(121-476). The CCM1 gene encoding Candida albicans cap methyltransferase (Ccm1p; 474 amino acids) was isolated from a C. albicans genomic library by selection for complementation of the conditional growth phenotype of S. cerevisiae abd1-ts mutants. Human cap methyltransferase was expressed in bacteria, purified, and characterized. Recombinant Hcm1p catalyzed quantitative S-adenosylmethionine-dependent conversion of GpppA-capped poly(A) to m7GpppA-capped poly(A). We identified by alanine-scanning mutagenesis eight amino acids (Asp-203, Gly-207, Asp-211, Asp-227, Arg-239, Tyr-289, Phe-291, and Phe-354) that are essential for human cap methyltransferase function in vivo. All eight residues are conserved in other cellular cap methyltransferases. Five of the mutant human proteins (D203A, R239A, Y289A, F291A, and F354A) were expressed in bacteria and found to be defective in cap methylation in vitro. Concordance of mutational effects on Hcm1p, Abd1p, and vaccinia capping enzyme underscores a conserved structural basis for cap methylation in DNA viruses, yeast, and metazoans. This is in contrast to the structural and mechanistic divergence of the RNA triphosphatase components of the yeast and metazoan capping systems. Nevertheless, we demonstrate that the entire three-component yeast capping apparatus, consisting of RNA 5'-triphosphatase (Cet1p), RNA guanylyltransferase (Ceg1p), and Abd1p could be replaced in vivo by the two-component mammalian apparatus consisting of a bifunctional triphosphatase-guanylyltransferase Mce1p and the methyltransferase Hcm1(121-476)p. Isogenic yeast strains with fungal versus mammalian capping systems should facilitate rational screens for antifungal drugs that target cap formation in vivo.

Expression and characterization of three tomato 1-aminocyclopropane-1-carboxylate oxidase cDNAs in yeast.

Heterologous expression in yeast has previously shown that the tomato cDNA LE-ACO1 encodes a functional 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACO) protein [Hamilton, A. J., Bouzayen, M. & Grierson, D. (1991) Proc. Natl Acad. Sci. USA 88, 7434-7437]. In the present work, full-length cDNAs encoding the two other members of the tomato ACO family (LE-ACO2 and LE-ACO3) were isolated and expressed in Saccharomyces cerevisiae. Analysis of the predicted amino acid sequences showed that the ACO1 and ACO3 proteins are highly similar (95%) while ACO2 is more divergent (89%). Yeast strains transformed with each of the three cDNAs were able to convert exogenous ACC to ethylene, the ACO1 strain exhibiting the highest activity in vivo and the ACO3 and ACO2 strains reaching 65% and 45% of ACO1 maximum activity, respectively. None of the ACO activities expressed in yeast required addition of ascorbate in vivo. ACO activities assayed in vitro revealed no significant differences between the three isoforms with regards to optimum temperature (29 degrees C), optimum pH (6.8-7.2), absolute dependence for ascorbate, Fe2+ and carbon dioxide, and inhibition by iron-chelating agents (1,10-phenanthroline and EDTA), Co2+ and free-radical scavengers (n-propyl gallate). However, differences were detected in the apparent Km values for ACC, the pI and the specific activity. The biochemical features that might explain the differences between the isoenzyme activities are discussed.

Human CPR (cell cycle progression restoration) genes impart a Far- phenotype on yeast cells.

Regulated cell cycle progression depends on the proper integration of growth control pathways with the basic cell cycle machinery. While many of the central molecules such as cyclins, CDKs, and CKIs are known, and many of the kinases and phosphatases that modify the CDKs have been identified, little is known about the additional layers of regulation that impinge upon these molecules. To identify new regulators of cell proliferation, we have selected for human and yeast cDNAs that when overexpressed were capable of specifically overcoming G1 arrest signals from the cell cycle branch of the mating pheromone pathway, while still maintaining the integrity of the transcriptional induction branch. We have identified 13 human CPR (cell cycle progression restoration) genes and 11 yeast OPY (overproduction-induced pheromone-resistant yeast) genes that specifically block the G1 arrest by mating pheromone. The CPR genes represent a variety of biochemical functions including a new cyclin, a tumor suppressor binding protein, chaperones, transcription factors, translation factors, RNA-binding proteins, as well as novel proteins. Several CPR genes require individual CLNs to promote pheromone resistance and those that require CLN3 increase the basal levels of Cln3 protein. Moreover, several of the yeast OPY genes have overlapping functions with the human CPR genes, indicating a possible conservation of roles.

The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast.

The budding yeast Cdc6 protein (Cdc6p) is essential for formation of pre-replicative complexes (pre-RCs) at origins of DNA replication. Regulation of pre-RC assembly plays a key role in making initiation of DNA synthesis dependent upon passage through mitosis and in limiting DNA replication to once per cell cycle. Cdc6p is normally only present at high levels during the G1 phase of the cell cycle. This is partly because the CDC6 gene is only transcribed during G1. In this article we show that rapid degradation of Cdc6p also contributes to this periodicity. Cdc6p degradation rates are regulated during the cell cycle, reaching a peak during late G1/early S phase. Removal of a 47-amino-acid domain near the N-terminus of Cdc6p prevents degradation of Cdc6p. Likewise, mutations in the Cdc4/34/53 pathway involved in ubiquitin-mediated degradation block proteolysis and genetic evidence is presented indicating that the N-terminus of Cdc6p interacts with the Cdc4/34/53 pathway, probably through Cdc4p. A stable Cdc6p mutant which is no longer degraded by the Cdc4/34/53 pathway is, none the less, fully functional. Constitutive overexpression of either wild-type or stable Cdc6p does not induce re-replication and does not induce assembly of pre-replicative complexes after DNA replication is complete.

Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT*3A, TPMT*2): mechanisms for the genetic polymorphism of TPMT activity.

TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including medications such as mercaptopurine and thioguanine. TPMT activity exhibits autosomal codominant genetic polymorphism, and patients inheriting TPMT deficiency are at high risk of potentially fatal hematopoietic toxicity. The most prevalent mutant alleles associated with TPMT deficiency in humans have been cloned and characterized (TPMT*2 and TPMT*3A), but the mechanisms for loss of catalytic activity have not been elucidated. In the present study, we established that erythrocyte TPMT activity was significantly related to the amount of TPMT protein on Western blots of erythrocytes from patients with TPMT activities of 0.4-23 units/ml pRBC (rs = 0.99; P < 0.001). Similarly, heterologous expression of wild-type (TPMT*1) and mutant (TPMT*2 and TPMT*3A) human cDNAs in yeast and COS-1 cells demonstrated comparable levels of TPMT mRNA but significantly lower TPMT protein with the mutant cDNAs. Rates of protein synthesis were comparable for wild-type and mutant proteins expressed in yeast and with in vitro translation in rabbit reticulocyte lysates. In contrast, pulse-chase experiments revealed significantly shorter degradation half-lives for TPMT*2 and TPMT*3A ( approximately 0.25 hr) compared with wild-type TPMT*1 (18 hr). The degradation of mutant proteins was impaired by ATP depletion and in yeast with mutant proteasomes (pre-1 strain) but unaffected by the lysosomal inhibitor chloroquine. These studies establish enhanced degradation of TPMT proteins encoded by TPMT*2 and TPMT*3A as mechanisms for lower TPMT protein and catalytic activity inherited by the predominant mutant alleles at the human TPMT locus.

Isolation of Cosmid and cDNA Clones in the Region Surrounding the BTK Gene at Xq21.3-q22

A regional physical and transcription map involving yeast artificial chromosomes (YACs), cosmids, and cDNAs has been constructed for Xq21.3-q22 around the gene BTK (formerly atk or BPK) defective in X-linked agammaglobulinemia (XLA). With a positional cloning strategy employing direct cDNA selection, novel cDNAs were found to cluster in the region of approximately 100 kb flanking the XLA and alpha-galactosidase A loci. While these widely expressed transcripts are in the area known to contain CpG islands, a less evolutionarily conserved gene, located more than 130 kb distal of DXS178, maps to cosmid clones that could not be digested with rare-cutting restriction enzymes. The presence of transcribed sequences flanking the BTK allowed us to investigate their involvement in complex XLA phenotypes. Southern blot analysis using cDNA clones isolated from this region permitted us to exclude a contiguous deletion syndrome as an underlying defect in three patients with XLA and associated growth hormone deficiency. A single XLA patient with torsion dystonia and cosegregating X-linked deafness has been found with a deletion in the 3' part of BTK extending centromerically into the flanking expressed sequence DXS1274E. This suggests a possible involvement of the DXS1274E in this phenotype. The GenBank accession numbers for novel cDNA sequences are as follows: DXS1269E (L20773), DXS1271E (UO1923), DXS1273E (UO1925), and DXS1274E (UO1922).